Method for stimulating growth in animals

ABSTRACT

The present invention relates to biologically-active copolymers and to compounds that can be safely used to cause the thymus of an animal or human to increase in size, thereby replenishing the T-lymphocyte population. When injected into an animal or human with an antigen, certain of the biologically-active copolymers cause immunosuppression to the antigen. 
     When injected into poultry, certain of the biologically-active copolymers modulate immune responsiveness. The biologically active copolymers are also useful as growth promoters for animals. Compounds within the biologically-active copolymers of the present invention are effective tumoricidal agents. At low concentration, certain of the biologically-active copolymers can cause dedifferentiated cells to differentiate. 
     The biologically-active copolymer of the present invention is a safe and non-toxic formulation that can effectively be administered to animals or humans. The biologically-active copolymer can be mixed in an oil and water emulsion and administered to an animal or human by injection or by oral administration.

Portions of this work were funded by a grant from the NationalInstitutes of Health, Grant Number ES03791.

1.0 CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.090,795, now abandoned, filed Aug. 28, 1987, which is acontinuation-in-part of U.S. application Ser. No. 872,111, nowabandoned, filed Jun. 6, 1986 which is a continuation-in-part of U.S.application Ser. No. 787,770, now abandoned, filed Oct. 15, 1985, whichis a continuation-in-part of U.S. application Ser. No. 745,917, nowabandoned, filed Jun. 18, 1985.

1.0 Cross-reference to Related Applications

2.0 Technical Field

3.0 Background of the Invention

3.1 Immune Stimulating Compounds

3.2 Autoimmune diseases

3.3 Growth Promoting Compounds

3.4 Antitumor Compounds

3.5 Ionophoric Compounds

4.0 Summary of the Invention

5.0 Detailed Description of the Preferred Embodiment

5.1 General Description

5.2 Chemical Structure

5.3 Biological Activities

5.4 Administration of the Biologically Active Copolymers

6.0 Brief Description of the Figures

7.0 Examples

7.1 Mast Cell Degranulation

7.2 Ionophore Activity

7.3 Histamine release vs. footpad swelling

7.4 Thymus Stimulation, Histology

7.5 Thymus Stimulation, Copolymer in Saline

7.6 Thymus Stimulation, Oil and Water Emulsion

7.7 Thymus Stimulation, General Observations

7.8 Growth Stimulation, Comparison of Copolymers

7.9 Growth Stimulation, Various Emulsions

7.10 Stimulation of Activity

7.11 Tumoricidal Activity

7.12 Differential Effect of Copolymers on Leukemic Cell Lines

7.13 Differentiation of Leukemic Cell Lines

7.14 Immunosuppressive Activity

7.15 Adoptive Transfer of Immunosuppression

7.16 Suppression of Experimental Allergic Encephalomyelitis

7.17 Immunomodulation in Poultry, Bursal Weight

7.18 Immunomodulation in Poultry, Bursal Morphology

7.19 Immunostimulation in Poultry

2.0 TECHNICAL FIELD

The present invention relates to compounds with a variety of biologicalactivities and more particularly, to a series of block copolymers thathave a wide variety of effects on living cells and organisms.

3.0 BACKGROUND OF THE INVENTION

The present invention is a class of compounds that has a wide variety ofprofound biological effects. Background material concerning several ofthese biological activities is discussed hereinbelow.

3.1 Immune Stimulating Compounds

The immune system is a highly complex system of cells and tissues thatrequires the cooperation of a large number of different cell types. Thesystems of the body that make up the immune system network are variouslycategorized as belonging to the hematopoietic system, thereticuloendothelial or phagocytic system and the lymphoid system.

The hematopoietic system is located in the bone marrow and isresponsible for supplying the various precursor and accessory cells ofthe immune systems. The reticuloendothelial system is made up of thephagocytic cells that are responsible for destroying or neutralizingforeign material that may enter the body. The lymphoid system is made upof lymphocytes, and is responsible for the overall regulation of theimmune system and for the production of antibodies.

The tissues of the lymphoid system are generally classified as thecentral tissues and the peripheral tissues. Two central lymphoid tissuesof mammals are bone marrow and thymus. In addition, fowl have a thirdcentral lymphoid organ, the bursa of Fabricius, which is critical to thedevelopment of the immunoglobulin-producing cells. It is thought thatthe mammals have a bursal equivalent associated with the intestinaltract. Lymph nodes, spleen, tonsils, intestinal lymphoid tissue (Peyer'spatches) and other collections of lymphocytes constitute the peripherallymphoid tissues.

In mammals, the bone marrow, if considered as a single tissue, is thelargest tissue of the body. In the average human adult the total weightof the bone marrow is about 3 kg. Marrow fills the central core ofnearly all bones. Bone marrow has three types of tissue; vasculartissue, adipose tissue and the tissue directed to hematopoiesis or bloodcell formation. The vascular tissue is the circulatory system thatsupplies nutrients and removes wastes from the actively growing cells.The hematopoietic tissue is responsible for the formation oferythrocytes, platelets, granulocytes and monocytes, and lymphocyteprecursors. Adipose tissue consists of fat cells which contribute littleto the function of the bone marrow.

The other central lymphoid tissue is the thymus; a bilobed organsituated in the anterior thoracic cage over the heart. In other species,the thymus may be distributed along the neck and thorax in severallobules.

Embryologically, the thymus emerges from the third and fourth branchialpouches. The human thymus is a fully developed organ at birth and weighs15 to 20 grams. By puberty it weighs 40 grams, after which it atrophiesor involutes becoming less significant structurally and functionally.Atrophy of the thymus with age is a characteristic of all species whichis associated with aging and the cessation of growth. The incidence ofage related diseases increases as the thymus shrinks andthymus-dependent immunity decreases. This age-associated decrease inthymic weight, called involution, is accompanied by changes in thethymic structure and a general decline in thymic function. Transientinvolution of the thymus may also occur as a consequence of a stress orinfection. Thymic involution may be controlled hormonally; castrationslows involution while injection of corticosteroid hormones acceleratesinvolution. Numerous studies have demonstrated that the thymicinvolution associated with increasing age parallels a reduction ofT-lymphocyte-mediated immunity and increased incidence of diseasesassociated with aging. Many diseases and treatments can accelerateinvolution of the thymus; virtually none are known to enhance growth ofthe thymus or reverse involution.

Anatomically, the thymus is a pouch of epithelial cells filled withlymphocytes, nourished and drained by the vascular and lymphatic systemsand innervated by the autonomic nerves. The epithelial cells and otherstructural cells divide the thymus into a complex assembly of continuouslobes, each of which is heavily laden with lymphocytes. The epithelialcells produce hormones which regulate some of the activities of thelymphocytes. The lymphocyte population is greatest in the cortex orouter portion of each lobule. The inner section, the medulla, has moreepithelial cells and fewer lymphocytes but the lymphocytes are moremature.

Lymphocytes can generally be classified as either T-lymphocytes or asB-lymphocytes. B-lymphocytes are responsible for the production ofantibodies (immunoglobulin) in response to a challenge by a particularantigen. T-lymphocytes are responsible for the general regulation of theimmune system and are also the principal mediators in cell-mediatedimmune responses. They also influence the proliferation of bone marrowcells and are probably involved in the growth and differentiation ofother organs as well.

All lymphocytes are ultimately derived from stem cells in bone marrow.These lymphocyte precursors are dispersed into the blood where theycourse through many organs. However, critical events take place in thethymus and bursa of Fabricius (or its mammalian equivalent) that imprintthe lymphocytes with special functions and that regulate the developmentinto either T or B-lymphocytes.

Life-Span studies of lymphocytes of most mammalian species dividelymphocytes into two fractions--those with a short span (mostly largelymphocytes) of 5 to 7 days and the small lymphocytes with a life spanmeasured in months or even years. The former are usually B-lymphocytesand the latter are usually T-lymphocytes.

B-lymphocytes respond to immunologic phenomena very differently from aT-lymphocyte in practically every instance. T-lymphocytes are formed inthe thymus from lymphoblasts that left the bone marrow. This maturationis expressed morphologically as a reduction in cell size to about 7 μmin diameter. The thymic cortex is rich in lymphocytes of all sizes.These thymocytes are not morphologically distinguishable fromlymphocytes in other tissues, but they are immature and antigenicallyidentifiable by the presence of several cell surface antigens includingthe φ, or T antigen, a distinctive surface marker antigen that separatesthe T-lymphocyte from the B-lymphocyte.

Enumeration of lymphocytes indicate that 65% to 85% of all lymphocytesin the blood are of the T type. Lymphocytes of the thoracic duct fluidare nearly 90% to 95% of the T variety and those in the Peyer's patchesor the gut are 50% to 65% T-lymphocytes. The T-lymphocyte population oflymph nodes, particularly in the deep cortical region, is high, but islow in the tonsil and the appendix.

When the T-lymphocyte contacts a recognizable antigen in the appropriatecontext, it passes through a phase of growth and cell division known aslymphocyte transformation to produce a large population of its own kind.The antigen must first be "processed" by macrophages and then presentedto T-lymphocytes.

T-lymphocytes are actually divided into several subsets and the rolethat they play in the immune system is complex. The T-lymphocyte isresponsible for the phenomenon known as the cell-mediated immuneresponse. In a cell-mediated immune response, the T-lymphocytes thatrecognize a cell-bound antigen begin producing and secreting a widevariety of proteins that affect the activity of other types of cells inthe immune system. These proteins include lymphokines that attract,activate and hold phagocytes at the site of the antigen and interferonsthat provide protection against virus infection.

The T-lymphocyte is also an important regulator of B lymphocytefunction. The antigen-exposed T-lymphocyte may have either of two directand opposite effects on B-lymphocytes depending on the subclass ofT-lymphocyte. The major subclasses are the helper cell and the other isthe suppressor cell. Helper T-lymphocytes are necessary for a complete Bcell response to T-lymphocyte-dependent antigens. T-lymphocyte dependentantigens tend to be the more complex antigens such as bacterialproteins, virus proteins and other large complex proteins in general.

Unlike helper T-lymphocytes, suppressor T-lymphocytes block thedevelopment of effector B and T-lymphocytes. Specific suppressorT-lymphocytes have now been demonstrated to play a large role intolerance to many proteins, both in antibody and cell-mediated immuneresponses. In addition, genetic unresponsiveness to some antigens is dueto the greater stimulation of suppressor T-lymphocytes than of helperT-lymphocytes by these antigens.

Thus, in the normal, healthy animal, the thymus is normally active onlyduring the early years of life. During these early years of thymicactivity, the thymus supplies the animal with the T-lymphocytes whichwill serve the animal for the rest of its life. In certain diseases,such as rheumatoid arthritis, the thymus may regain some activity duringadult life. This demonstrates that the adult thymus retains capacity tofunction and that involution is not necessarily permanent. At leastpartial function might be restored if the appropriate agents wereavailable.

Acquired T-lymphocyte deficiency diseases of adults are characterized bya depletion of circulating T-lymphocytes. The symptoms expressed inthese diseases include an inability to mount a cell mediated immuneresponse in response to an antigen challenge. An example of an acquiredT-lymphocyte deficiency disease is acquired immune deficiency syndromeor AIDS.

AIDS is a disease caused by the human T-lymphocyte lymphotrophic virus(formerly LAV or HTLV-III; currently designated HIV). The virusspecifically attacks T-4 helper lymphocytes, a subgroup of T-lymphocytesthat plays a major role in defending the body against infectiousdiseases. Depletion of this subset of lymphocytes is manifested by anincreased incidence of opportunistic infections like pneumocystiscarinii and certain cancers. More specifically, the virus enters theT-lymphocyte and incorporates viral encoded DNA into the DNA of the hostT-lymphocyte. As long as the infected T-lymphocyte remains inactivated,the virus will quietly remain in the DNA of the host cell. This will notkill the cell but may impair its function. When the infectedT-lymphocytes are activated by stimuli such as a specific antigen, theviral DNA in the host DNA is expressed and produces new viral particles.The host T-lymphocyte is then killed and lysed, releasing new viralparticles that can invade and kill other T-lymphocytes. The loss of T-4lymphocytes is profound and occurs even faster than can be accounted forby direct viral killing of the cells. This has led some investigators topostulate that the infection somehow shuts off the production of T-4lymphocytes. In any case, the thymus in the normal adult is no longerfunctioning and the killed T-lymphocytes cannot be replaced leaving thepatient vulnerable to subsequent infections. Especially striking arerecent studies of the thymuses of deceased AIDS patients ranging in agefrom 10 months to 42 years. AIDS victims have profound thymicinvolution; much more extensive than in age-matched patients who died ofother causes.

The cure of a person with AIDS will probably require one agent toeliminate the virus and other agents to cause the body to replace Tcells that have been killed by the virus. The first step is to eliminatethe AIDS virus from the patient. This will have to be supported by othertherapies to induce restoration of immune function. Studies to date withmacrophage activating agents, interferon inducers and lymphokines havebeen disappointing, possibly because their targets, T-lymphocytes, donot exist in sufficient numbers. Interleukin 2 restores the function ofone subset of non T-cells (natural killer cells) but has no effect on ahost of other serious defects. More drastic measures can be performed.One potential method of restoring the immune system is by transplantingbone marrow from healthy donors. However, this is a dangerous procedure.It may produce lethal graft versus host disease unless the patient'sdonor is an identical twin.

Another area where there is a need to re-establish not only the immunesystem, but also the hematopoietic system, is in total body irradiationfor treatment of leukemia. When a patient undergoes high dose total bodyirradiation, the entire immune system is destroyed. The usual treatmentafter the irradiation is to perform a bone marrow transplant with marrowfrom a close relative. If the transplant is successful, the new marrowwill produce new cells, thereby restoring both red blood cells and whiteblood cells to the body. However, this is a dangerous treatment that issuccessful in only a fraction of the cases. Localized radiation oftumors and several types of chemotherapy also produce suppression ofT-cell mediated immunity.

What is needed is a safe and effective method of re-establishingT-lymphocyte function. One method of re-establishing T-lymphocytefunction is by treating existing T-lymphocytes so that they resume theirnormal immune functions. Agents that have been shown to be effective incertain situations in stimulating T-lymphocytes include macrophageactivating factors, interferon inducing agents, lymphokines andcytokines. However, in a disease such as AIDS or in the case ofirradiation in which the T-lymphocyte population has been destroyed,this type of treatment is not effective because the number ofT-lymphocytes is severely depleted. In these cases, an effective methodof causing the thymus to produce new T-lymphocytes would be thetreatment of choice. However, to date, there is no effective treatmentthat will cause the thymus to reverse the process of involution andproduce new T-lymphocytes.

While restoration or enhancement of the immune system in humans isapplied on a case by case basis, immune modulation of livestock ispreferably applied to entire herds of cattle, sheep and pigs, and toentire flocks of chickens. In particular, the need for immune modulationof poultry has become increasingly necessary as the demand forconsumable poultry and the spread of poultry disease escalates.

Poultry disease such as Coccidiosis and Infectious Bursal Disease havebecome widespread among commercial poultry institutions and areresponsible for the death of millions of chickens each year. Vaccineshave been developed against many of the poultry diseases, but someinoculated flocks fail to gain immunity and, upon exposure, contract thedisease and die. Even in the absence of such epidemics, significantlosses are suffered from opportunistic infections by bacteria and fungiwhich rarely cause disease in immune competent chickens.

Normally chickens are inoculated at an early age to ensure that theyreceive adequate vaccination before they become infected by exposure toother birds carrying disease. However, most chicks inoculated at such anearly age have inadequate immune responsiveness because humoral immunemechanisms have not fully developed.

What is needed in poultry is the precocious development of humoralimmunoresponsiveness in young chickens so that vaccinated chicks candevelop protective immunity to pathogens before they become infected.

3.2 Autoimmune Diseases

Autoimmune diseases are characterized by the development of an immunereaction to self components. Normally, tissues of the body are protectedfrom attack by the immune system; in autoimmune diseases there is abreakdown of the self-protection mechanisms and an immune responsedirected to various components of the body ensues. Autoimmune diseasesare for the most part chronic and require life long therapy. The numberof recognized autoimmune diseases is large and consists of a continuumranging from diseases affecting a single organ system to those affectingseveral organ systems. With increased understanding of the molecularbasis of disease processes, many more diseases will likely be found tohave an autoimmune component. Specific examples of autoimmune diseasesare presented below.

SPECTRUM OF AUTOIMMUNE DISEASES

Organ Specific

Hashimoto's thyroiditis

Graves' disease

Addison's disease

Juvenile diabetes (Type I)

Myasthenia gravis

Pemphigus vulgaris

Sympathetic opthalmia

Multiple sclerosis

Autoimmunehemolytic anemia

Active chronic hepatitis

Rheumatoid arthritis

Non-organ specific

Systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an inflammatory, multisystemdisease characterized clinically as a relapsing disease of acute orinsidious onset that may involve any organ in the body. Clinically,symptoms are due to disease affecting the skin, kidneys, serosalmembranes, joints and heart. Anatomically, all sites have in commonvascular lesions with fibrinoid deposits and immunologically, thedisease involves antibodies of autoimmune origin, especially antinuclearantibodies (ANA). The ANA are directed against both DNA and RNA.Autoantibody development appears to be multifactorial in origin,involving genetic, hormonal, immunologic and environmental factors.

The morphologic changes seen in organs result from the formation ofcirculating immune complexes and their deposition in a variety oftissues. Although many organs can be affected, some are affected morethan others. Lesion of joints, the kidneys, heart, and serous membranesare responsible for most of the clinical signs. The course of SLE isextremely variable and unpredictable. An acute onset with progressivedownhill course to death within months can occur. The usual coursehowever, is characterized by flare-ups and remissions spanning a periodof years or even decades. It usually arises in the second or thirddecades of life, but may become manifest at any age.

Acute attacks are usually treated by adrenocortical steroids orimmunosuppressive drugs. These drugs often control the acutemanifestations. With cessation of therapy the disease usuallyreexacerbates. The prognosis has improved in the recent past;approximately 70 to 80% of patients are alive 5 years after the onset ofillness and 60% at 10 years. Lifelong therapy is required to control thedisease.

At one time SLE was considered to be a fairly rare disease. Bettermethods of diagnosis and increased awareness that it may be mild andinsidious have made it evident that its prevalence may be as high as 1case per 10,000 population. There is a strong femalepreponderance--about 10 to 1.

Rheumatoid arthritis is a systemic, chronic, inflammatory disease thataffects principally the joints and sometimes many other organs andtissues throughout the body. The disease is characterized by anonsuppurative proliferative synovitis, which in time leads to thedestruction of articular cartilage and progressive disabling arthritis.The disease is caused by persistent and self-perpetuating inflammationresulting from immunologic processes taking place in the joints. As isthe case with most autoimmune diseases, the trigger that initiates theimmune reaction remains unidentified. Both humoral and cell mediatedimmune responses are involved in the pathogenesis of rheumatoidarthritis. The majority of patients have elevated levels of serumimmunoglobulins and essentially all patients have an antibody calledrheumatoid factor (RF) directed against a component of another antibodyclass.

The key event in the pathogenesis of the arthritis is the formation ofantibodies directed against other self antibodies. Why these antibodiesare formed is unknown at present. It has been suggested that the processis initiated by the formation of antibodies or immunoglobulins againstan unknown antigen, possibly an infectious agent. When the antibodiescombine with the antigen, conformational changes occur in a portion ofthe antibody molecule creating new antigenic determinants. Theappearance of new determinants evokes an antibody response against theantibody molecule and results in the formation of anti-immunoglobulinantibodies or rheumatoid factor. T cells may also be involved in thepathogenesis of rheumatoid arthritis. A large number of T cells arefound in the synovial membrane, outnumbering B cells and plasma cells.Additionally, procedures to decrease the population of T cells (such asdraining the thoracic duct), result in remission of symptoms.

The most destructive effects of rheumatoid arthritis are seen in thejoints. Classically, it produces symmetric arthritis, which principallyaffects the small joints of the hands and feet, ankles, knees, wrists,elbows, shoulders, temporo-mandibular joints and sometimes the joints ofthe vertebral column. The clinical course is highly variable. Afterapproximately 10 years, the disease in about 50% of the patients becomesstabilized or may even regress. Most of the remainder pursue a chronic,remitting, relapsing course. After 10 to 15 years, approximately 10% ofpatients become permanently and severely crippled. The disease usuallyhas its onset in young adults but may begin at any age and is 3 to 5times more common in women than in men.

Rheumatoid arthritis is a very common disease and is variously reported(depending on diagnostic criteria) to affect 0.5 to 3.8% of women and0.1 to 1.3% of men in the United States.

Multiple sclerosis is another disease that is thought to be caused byautoimmune mechanisms. The cause of multiple sclerosis is unknown butseems to be multifactorial. Susceptibility or resistance may begenetically determined; something in the environment interacts with thehuman host at the proper age to cause biochemical and structural lesionsin the central nervous system. The systemic immune response and theresponse of the central nervous system become involved. Although thecause and pathogenesis of multiple sclerosis are unknown, it is widelybelieved that immune abnormalities are somehow related to the disease.Three possible mechanisms have been postulated: infection, autoimmunity,and a combination of the two. Suppression or modulation of the immuneresponses may be the key.

The graphic distribution of multiple sclerosis indicates that thedisease is acquired from an environmental factor. Approximately 200studies of the geographic distribution of multiple sclerosis have beenconducted and have shown that regions of high prevalence (30 to 80 casesper 100,000 population) in northern Europe between 65 and 45 degreesnorth latitude and in the northern United States and southern Canada, aswell as in southern Australia and New Zealand. In contrast, regions oflow risk, including most of Asia and Africa, have a prevalence of 5 orfewer cases per 100,000.

Myasthenia gravis is an autoimmune disorder caused by antibodiesdirected against the acetylcholine receptor of skeletal muscle. Presentinformation indicates at least three mechanisms whereby acetylcholinereceptor antibody may interfere with neuromuscular transmission and thusinduce myasthenia gravis. Acetylcholine receptor antibody may interfere(directly or indirectly) with acetylcholine receptor function. In bothexperimental allergic myasthenia gravis and human myasthenia gravis, theextent of acetylcholine receptor loss parallels the clinical severity ofthe disease, suggesting that acetylcholine receptor antibody-inducedacceleration of acetylcholine receptor degradation is important in thedevelopment of myasthenia gravis. Complement-mediated destruction of thepostsynaptic region is the third possible cause. Other disorders,especially those presumed to be autoimmune in origin, can occur inassociation with myasthenia gravis. Thyroid disease, rheumatoidarthritis, systemic lupus erythematosus, and pernicious anemia all occurmore commonly with myasthenia gravis than would be expected by chance.

The prevalence of myasthenia gravis in the United States is one per20,000.

The foundation of therapy of autoimmune diseases is treatment withimmunosuppressive agents. The basis for this therapy is attenuation ofthe self-directed immune response with the primary aim being to controlsymptoms of the particular disease. The drugs utilized to achieve thisaim are far from satisfactory, in that adverse side effects are numerousand control of the disease is many times difficult to achieve. Theproblem is compounded by the chronicity of the disease with effectivetherapy becoming more difficult with time. An indication of the severityof particular diseases is seen in the willingness to accept greaterrisks associated with therapy as the disease progresses. Currentlyavailable therapy is distinctly non-selective in nature, having broadeffects on both the humoral and cell mediated arms of the immune system.This lack of specificity can limit the effectiveness of certaintherapeutic regimens. The main groups of chemical immunosuppressives arealkylating agents, antimetabolites, corticosteroids, and antibiotics,each will be discussed briefly.

The corticosteroids, also called adrenocorticosteroids, are fat-likecompounds produced by the outer layer or cortex, of the adrenal gland.The adrenal cortex is an organ of homeostasis influencing the functionof most systems in the body. It is responsible for adaptation of thebody to a changing environment. Therapeutic use of the corticosteroidsfor autoimmune disease is based on their two primary effects on theimmune system, anti-inflammatory action and destruction of susceptiblelymphocytes. They also effect a redistribution of lymphocytes fromperipheral blood back to the bone marrow. The use of corticosteroids isnot without adverse side effects however, particularly during the courseof life-long treatment which is required for many of the autoimmunediseases. Major side effects of steroids are:

1. Cushing syndrome

2. Muscle atrophy

3. Osteoporosis

4. Steroid induced diabetes

5. Atrophy of the adrenal glands

6. Interference with growth

7. Susceptibility to infections

8. Aseptic bone necrosis

9. Cataract development

10. Gastric ulcer

11. Steroid psychosis

12. Skin alterations

13. Nervous state accompanied by insomnia

Attempts to minimize side effects incorporate alternate day or lessfrequent dosage regimens.

A recently developed immunosuppressive agent is the antibioticcyclosporin A. The antibiotic has greatest activity against T cells anddoes not seem to have much direct effect on B cells. The drug is beingevaluated for the treatment of autoimmune diseases for which it showssome promise. Side effects include hair growth, mild water retention,renal toxicity, and, in older patients, nervous system disorderssymptoms have been observed.

Other drugs are used alone or in combination with those listed above andinclude gold salts and antimalarials, such as chloroquine. Another classof drugs, the non-steroidal anti-inflammatory drugs are used extensivelyin arthritis. These drugs provide analgesia at low doses and areanti-inflammatory after repeated administration of high doses.Nonsteroidal anti-inflammatory drugs all act rapidly and their clinicaleffects decline promptly after cessation of therapy. They do not preventthe progression of rheumatoid arthritis and do not induce remissions.Immunostimulants, such as levamisol have also been used in manyautoimmune diseases but side effects have generally limited their use.

3.3 Growth Promoting Compounds

With an ever-increasing world demand for food, there is constantpressure to increase the rate of production of food. In the early1950's, researchers unexpectedly discovered that an antibioticingredient in chicken mash was a "growth factor." The findingdrastically changed the nation's livestock and poultry production andwas an economic boon for pharmaceutical companies. Feed animals are nowraised under highly controlled conditions and receive specialized feedwith a variety of growth promoting additives.

Routine antibiotic administration to animals has become almost universalsince the discovery that the addition of small amounts of antibioticssuch as penicillin, tetracycline and sulfamethazine, to animal feedincreases the growth of pigs and cattle. In 1979, about 70% of the beefcattle and veal, 90% of the swine, and virtually 100% of broilers rearedin the United States consumed antibiotics as part of their daily feed.This use, accounting for nearly 40% of antibiotics sold in the UnitedStates, is estimated to save consumers $3.5 billion a year in foodcosts.

Animals raised under modern conditions optimized for growth promotionreceive rations containing high proportions of protein, usually in theform of soybean or cottonseed meal, and high percentages of grains suchas corn or milo, a type of sorghum. Feed additives which have been usedinclude such hormones as diethyl-stilbesterol, which also increases therate of weight gain, and tranquilizers that prevent the effects of thestress brought on by confinement conditions from causing disease orweight loss.

Cattle ordinarily require 10 pounds of feed to produce one pound ofweight gain. Under optimal growth promoting conditions and with enrichedfeed they gain one pound with only 6 pounds of feed.

Modern farming has greatly reduced the labor required to raise farmanimals. In broiler chicken raising, where intensive methods have hadthe most dramatic effect, it took 16 hours of labor to raise a flock of100 broilers in 1945; in 1970 that figure was reduced to 1.4 laborhours, in part because of the use of automated confinement facilitiesand associated advances in breeding and nutrition.

Although hormones and antibiotics have greatly increased the rate ofgrowth of food animals, the use of such additives has not been withoutproblems. One of the hormones that is commonly used as a growthstimulant, diethyl- stilbesterol or DES, has been shown to be acarcinogen and has been banned from further use in most countries.

When antibiotics are mixed in animal feed, the compounds are spreadthroughout the environment exposing microorganisms to the antibiotics.The constant exposure of the microorganisms to antibiotics putsbiological pressure on the microorganisms to develop a resistance to theantibiotics. This can result in a microorganism that is resistant toantibiotics and causes especially severe and difficult to treatinfections.

An antibiotic-resistant microorganism is potentially a serious pathogenbecause it is difficult to control. If the organism causes an infectionin an animal or in man, the infection may not be controlled withconventional antibiotics. If the infection is serious, there may not betime to determine which antibiotics are effective against the infectingbacteria. The problem has been especially serious when antibioticresistant organisms in meat are consumed by people who themselves takeantibiotics for treatment of disease. Antibiotics inhibit many of thenormal microorganisms in the respiratory and gastrointestinal tracts.This allows the resistant one to proliferate rapidly and produce moreserious disease. The combination of antibiotic resistant organisms fromfood and ineffective antibiotic treatment of people has caused most ofthe deaths due to salmonella food poisoning reported in the UnitedStates in the past several years.

As a result of the increasing appearance of antibiotic resistantbacteria in feed lots and several serious epidemics caused by antibioticresistant bacteria, there is increasing governmental pressure to ban theuse of antibiotics in animal feed. Consequently, there is an immediateand increasing need for new, safe and effective growth stimulators offarm animals.

3.4 Antitumor Compounds

Malignant, or cancerous, tumors are defined by their invasion of localtissue and their ability to spread or metastasize to other parts of thebody. The incidence of tumors is high; it is the second leading cause ofdeath in both children and adults. A malignant tumor, by definition,always kills (unless treated) because of its invasive and metastaticcharacteristics. The tumor grows locally by encroachment into the normaltissues surrounding it. The tumor spreads to distant sites by thebreaking off of malignant cells. These cells then move through the bloodand lymphatic systems, attach themselves, and begin to grow as newcolonies.

The factors controlling tumor growth are poorly understood. Tumors inlaboratory animals may be transplanted to a second host using only asingle tumor cell. This facility suggest that only one normal cell needbecome transformed (cancerous) for tumor growth to begin. It is thought,however, that many transformed cells die or remain latent or dormant forextended periods before successful tumor growth is established. Tumorshave been experimentally induced in animals by chemical, physical, andviral agents, and by radiation and chronic irritation.

Leukemia is a term given to tumors of the blood-forming organs. Theacute and chronic leukemias, together with the other types of tumors ofthe blood, bone marrow cells (myelomas), and lymph tissue (lymphomas),cause about 10% of all cancer deaths and about 50% of all cancer deathsin children and adults less than 30 years old. At least 4 million peoplenow living are expected to die from these forms of cancer, assumingthere are no major advancements made in the treatment of these diseases.

Conventional treatment regimens for leukemia and for other tumorsinclude radiation and drugs or a combination of both. In addition toradiation, the following drugs, usually in combinations with each other,are often used to treat acute leukemias: vincristine, prednisone,methotrexate, mercaptopurine, cyclophosphamide, and cytarabine. Inchronic leukemia, for example, busulfan, melphalan, and chlorambucil canbe used in combination. All of the conventional anti-cancer drugs arehighly toxic and tend to make patients quite ill while undergoingtreatment. Vigorous therapy is based on the premise that unless everyleukemic cell is destroyed, the residual cells will multiply and cause arelapse.

Most of the conventional chemotherapeutic drugs that are being used intumor therapy do not specifically kill tumor cells. Reliance is placedon the fact that, in most cancers, the cancerous cells grow faster thannormal cells and will therefore utilize more of the toxicchemotherapeutic drug thereby specifically killing the cancer cell.Administration of the conventional chemotherapeutic drugs requirescareful attention to the amount and concentration of the drug orcombination of drugs so that the cancer cells will be killed but normalcells will survive. For this reason, it is difficult to kill allcancerous cells by conventional chemotherapy.

What is needed are compounds that will specifically and completely killcancerous cells while not effecting normal cells. Ideally, the newcompounds would take advantage of physical characteristics inherent onlyin the tumor cell. For example, a tumor cell may be more sensitive thannormal cells to changes in ion concentrations within the cell. If acompound could detrimentally vary the internal ion concentrations of thetumor cells, then the compound could specifically kill the tumor cellwhile not adversely affecting normal cells.

3.5 Ionophoric Compounds

Ionophores are defined as substances capable of interactingstoichoimetrically with metal ions so as to transport the ions across ahydrophobic barrier such as a cell membrane.

It has been generally accepted that cell membranes consist of aphospholipid bilayer interspersed with globular protein molecules. Thehydrophilic phosphate portions of the phospholipid are oriented at theouter edges of the membrane while the hydrophobic lipid portions facetoward the center. The cell membrane is selectively permeable and willpermit the entry of water, certain nutrients and essential metal ions topass freely into the cell when needed. However, due to the double layerof nonpolar lipids in its center, the membrane is normally impermeableto highly polar molecules.

Different ionophores often have an affinity for one ion or one group ofions over another. The ions most commonly transported across cellmembranes include Na⁺, K⁺, Li⁺, Rb⁺, Cs⁺, Ca⁺², Mg⁺², Ba⁺², Cu⁺², Fe⁺²,Ni⁺², and Zn⁺². For example, the negatively charged fungal antibioticionophore A23187 selectively forms an electrically neutral "encountercomplex" with positively charged calcium ions. This hydrophobic moleculeis capable of moving across a number of different cell membranes, andonce the complex enters the cell, the calcium ion is released. Thisincrease in intracellular free calcium has been shown to stimulate thesecretion of a variety of substances such as histamine from rodent mastcells and human basophils, amylase and insulin from the pancreas, thehormone vasopressin from the pituitary, the neurotransmitter dopaminefrom neurons, seratonin from platelets, and catecholamines from adrenalglands. In addition, the A23187 calcium ionophore has been shown toactivate sea urchin eggs.

With an ever-increasing world demand for food, there is constantpressure to increase the efficiency of production of food. Ruminantnutritionists have long sought means to manipulate and improve theefficiency of ruminal fermentation. Dietary manipulation was initiallyused to achieve this goal, but during the last decade a number of activeantibiotic compounds, produced by various strains of Streptomyces, werediscovered which improve metabolic efficiency. Although originallyadministered to poultry as anticoccidials, these carboxylic polyetherantibiotic compounds, including monensin, lasalosid, salinomycin andnarasin, have been found to exhibit ionophoric activity.

Since their discovery, antibiotic ionophores have been used extensivelyas feed additives to increase the efficiency of the production ofpoultry and ruminants. Studies have indicated that, when ionophores areadded to feed, the growth of pathogens and other microorganisms withinthe digestive tract is inhibited, thus enhancing the efficientutilization of nutrients in the feed.

The various antibiotic ionophores appear to improve the efficiency ofconversion from grain to meat by increasing the efficiency of metabolismin the rumen, improving nitrogen metabolism, and by retarding feedlotdisorders such as chronic lactic acidosis and bloat. These effects arecaused by a shift in the rumen microflora from bacteria less efficientin fermenting ingested feed to more bacteria that are more efficient.The change in rumen microflora population is brought about by adifferential susceptibility of the bacteria to ion flux across theirmembranes. This influx of ions causes the bacterial cells to swell andburst.

Although antibiotic ionophores have greatly increased the efficiency ofproduction of feed animals, the use of such additives has not beenwithout problems. When antibiotics are mixed in animal feed, thecompounds are spread throughout the environment exposing microorganismsto the antibiotics. The constant exposure of the microorganisms toantibiotics causes a resistance to antibiotics which in turn causesinfections which are especially severe and difficult to treat.

An antibiotic-resistant microorganism is potentially a serious pathogenbecause it is difficult to control. If the organism causes an infectionin an animal or in man, the infection may not be controlled withconventional antibiotics. If the infection is serious, there may not betime to determine which antibiotics are effective against the infectingbacteria. The problem has been especially serious when antibioticresistant organisms in meat are consumed by people who themselves takeantibiotics for treatment of disease. Antibiotics inhibit many of thenormal microorganisms in the respiratory and gastrointestinal tracts.This allows the resistant ones to proliferate rapidly and produce moreserious disease. The combination of antibiotic resistant organisms fromfood and ineffective antibiotic treatment of people has caused most ofthe deaths due to salmonella food poisoning reported in the UntiedStates in the past several years.

It is believed that there is currently no single class of compounds thatpossess such a wide range of biological activities. Such a class ofcompounds would provide a new and powerful arsenal for the treatment ofdisease and for increasing the worlds food supply.

4. SUMMARY OF THE INVENTION

In accordance with the present invention, a new class ofbiologically-active copolymers is provided which is capable of effectingbiological systems in many different ways. The biologically activecopolymers of the present invention are capable of stimulating thegrowth of an organism, stimulating the motor activity of an organism,stimulating the production of T-cells in the thymus, peripheral lymphoidtissue, and bone marrow cells of an animal, and stimulating immuneresponsiveness of poultry.

The biologically active copolymers of the present invention also have awide variety of effects on individual cells. These compounds haveionophoric activity, i.e., they cause certain ions to be transportedacross cell membranes. The compounds can cause non-cytolytic mast celldegranulation with subsequent histamine release. In addition, it hasbeen found that certain members of this class of biologically-activecopolymers are capable of specifically killing certain cancer celllines. The biologically active copolymers are also effective againstcertain microorganisms.

The biologically-active copolymers of the present invention can beadministered orally to animals to provide specific effects on certainmicroorganisms that reside in the gut of the animal. For example,certain biologically-active copolymers can be administered to chickensto kill various species of coccidia that cause coccidiosis.

The biologically-active copolymers can also be added to cattle feed toeffect a change in the population of microorganisms normally resident inthe rumen. Under normal conditions, the microorganisms digest thecellulose that is eaten by the cattle to the end-product methane.Methane is essentially unusable by the cattle. By administering thebiologically-active copolymers of the present invention orally to thecattle, the copolymer differentially affects the rumen microorganisms sothat there is a shift in the rumen population of microorganismsresulting in an increase in proprionic acid production and a decrease inlactic acid and methane. Cattle are capable of using proprionate intheir own metabolism thereby increasing the efficiency of foodconversion.

Biologic effects of the copolymers of the present invention vary withthe structure of the polymer. The ability to modify the structure ofthese polymers to optimize particular biologic effects provides thepotential to design synthetic compounds with a precision and ease notpossible in other systems.

The biologically-active copolymer of the present invention comprises acopolymer of polyoxyethylene (POE) which is hydrophilic andpolyoxypropylene (POP) which is hydrophobic. The block copolymer isbuilt on a tetrafunctional ethylenediamine initiator. In the preferredembodiment of the biologically-active copolymers of the presentinvention, the block copolymers that comprise the biologically-activecopolymers of the present invention have the following general formulas:##STR1## wherein: the mean aggregate molecular weight of the hydrophobeportion of the octablock copolymer consisting of polyoxypropylene (C₃ H₆O)_(b) (POP) is between approximately 5000 and 7000 daltons;

a is a number such that the hydrophile portion represented bypolyoxyethylene (C₂ H₄ O)_(a) (POE) constitutes between approximately10% and 40% of the total molecular weight of the compound; and

b is a number such that the polyoxypropylene (C₃ H₆ O)_(b) (POP) portionof the total molecular weight of the octablock copolymer constitutesbetween approximately 60% and 90% of the compound.

In another embodiment of the biologically-active copolymer of thepresent invention, the block copolymer comprises a polymer ofhydrophilic polyoxyethylene (POE) built on an ethylene diamineinitiator. Polymers of hydrophobic polyoxypropylene (POP) are then addedto block of hydrophilic polyoxyethylene (POE). This results in anoctablock copolymer with the following general formula: ##STR2##wherein: a is a number such that the hydrophile portion represented bypolyoxyethylene (C₂ H₄ O)_(a) (POE) constitutes between approximately10% to 40% of the total molecular weight of the compound;

the mean aggregate molecular weight of the hydrophobe portion of theoctablock copolymer consisting of polyoxypropylene (C₃ H₆ O)_(b) (POP)is between approximately 5000 and 7000 daltons; and

b is a number such that the polyoxypropylene (C₃ H₆ O)_(b) (POP) portionof the total molecular weight of the octablock copolymer constitutesbetween approximately 60% and 90% of the compound.

The biologically-active copolymer of the present invention is usuallyadministered by a subcutaneous, intravenous, or intramuscular injectionof an effective amount of the copolymer into an animal or human. Thebiologically-active copolymer of the present invention can be takenorally if it is desired that the copolymer have an effect on alimentarycanal microorganisms. Normally, very little of the copolymer is absorbedfrom the alimentary canal.

Accordingly, it is an object of the present invention to providecompounds that have a wide variety of biological activities.

Another object of the present invention is to provide compounds that canstimulate the T-cell immune system.

Another object of the present invention is to provide a compound thatwill stimulate the growth of the thymus in an adult animal.

Another object of the present invention is to provide compounds that canstimulate the production of bone marrow cells.

Another object of the present invention is to provide a compound thatcan stimulate precocious immune competence in poultry.

A further object of the present invention is to stimulate bone marrowand enhance recovery from radiation or other insults toxic to the bonemarrow.

Another object of the present invention is to provide compounds that canaccelerate and prolong growth.

Yet another object of the present invention is to provide compounds thatcan stimulate motor activity in animals and humans.

Another object of the present invention is to provide compounds thathave ionophore activity.

Another object of the present invention is to provide compounds that cancause non-cytolytic mast cell degranulation.

Another object of the present invention is to provide compounds that canspecifically kill certain tumor cell lines.

Another object of the present invention is to provide compounds thatwill cause a dedifferentiated cell to revert to a differentiated cell.

Another object of the present invention is to provide a compound thatcan increase the growth of an animal when taken orally.

Another object of the present invention is to provide compounds that cankill microorganisms that reside in the gut.

Another object of the present invention is to specificallyimmunosuppress an animal against an antigen or a hapten.

Yet another object of the present invention is to provide compounds thatare capable of altering the metabolism of ruminant animals so that theefficiency of feed conversion is increased.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiment and the appended claims.

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 5.1 GeneralDescription

The present invention comprises a class of biologically activecopolymers which have a wide variety of biological functions which areuseful in treating various pathological conditions in both humans andanimals and which can also be used to stimulate immunity and to increasethe efficiency of food production.

The biologically-active copolymers of the present invention comprise asurface active compound with hydrophobic segments and a small proportionof hydrophile that is built upon an initiator compound. The initiatorcompound typically has one or more active hydrogens upon which thehydrophobic or hydrophilic polymer units are condensed. Compounds thatcan be used as initiators in producing the biologically activecopolymers of the present invention include, but are not limited to,methylamine, ethylamine, propylamine, butylamine, amylamine, hexylamine,aniline, the alkylene polyamines, especially aliphatic primary diaminessuch as ethylenediamine, propylene diamine, diethylenetriamine,triethylenetetramine, tetraethylenepentamine, hexamethylene-diamine,phenylenediamine, and the like. Alkanolamines such as monoethanolamine,diethanolamine, triethanolamine, isopropanolamine, tri(t-propanol)amine,2-amino-1-butanol, N-butyl-di(2-propanol)amine can also be used asinitiators. Furthermore, heterocyclic compounds containing a heteronitrogen atom can be employed, such as piperazine, 2 methylpiperazine,2,5-dimethylpiperazine, imidazimidazole, pyrazolidine, pyrazolidone,hydantoin, dimethyl hydantoin and the like. Hydroxyl amine and thehydroxylamine derivatives and aminophenol and aminophenol derivativescan also be used as initiators to produce the biologically activecopolymers of the present invention. Other compounds that can be used asinitiators to produce the biologically active copolymers of the presentinvention include glycerol, 1,4 butanediol, 1,3 propylene glycol,diethylene glycol, triethylene glycol and penterythritol.

The compositions of this invention are surface active mixtures ofconjugated polyoxypropylenepolyoxyethylene compounds based on thereactive hydrogen compounds wherein chains of oxypropylene groups havinga defined molecular weight are attached to the initiator of the reactivehydrogen compound at the sites of the reactive hydrogen atoms andwherein chains of oxyethylene groups are then attached to the ends ofthe oxypropylene chains. Alternatively, the chains of oxyethylene groupshaving a defined molecular weight can be attached to the initiator ofthe reactive hydrogen compound at the sites of the reactive hydrogenatoms and then chains of oxypropylene groups can be attached to the endsof the oxyethylene chains.

The compositions are prepared by condensing either ethylene oxide orpropylene oxide with the reactive hydrogen on the initiator compound.After the first block of monomer units have been added to the initiatormolecule, a second block of either propylene oxide or ethylene oxide iscondensed with the reactive hydrogen on the end of the first block. Itis to be understood that butylene oxide can be substituted, either allor part, for propylene oxide in the biologically active copolymers ofthe present invention.

It is to be noted that it is not necessary to use pure propylene oxidein producing the oxypropylene chains of the biologically activecopolymers of the present invention. Small amounts, for example, up to 5weight percent, of ethylene oxide can be included in the propylene oxideemployed to prepare the hydrophobic reactive hydrogen compound-propyleneoxide condensate. Likewise, the ethylene oxide condensed with thehydrophobic propylene oxide-reactive hydrogen compound condensate canalso contain small amounts, such as up to about 5 weight percent, ofpropylene oxide.

It is further to be noted that when molecular weight is stated in thisspecification and claims, unless otherwise noted, there is meant theaverage theoretical hydrophobe molecular weight which equals the totalof the grams of the propylene oxide employed per mole of reactivehydrogen compound. It is well recognized in the field of alkylene oxidechemistry that the polyoxyalkylene compositions one obtains bycondensing an alkylene oxide with a reactive hydrogen compound areactually mixtures of compounds rather than a single molecular compound.The mixture contains closely related homologues wherein the statisticalaverage number of oxyalkylene groups equals the number of moles of thealkylene oxide employed and the individual members in the mixturecontain varying numbers of oxyalkylene groups. Thus, the compositions ofthis invention are "mixtures" of compounds which are defined bymolecular weight of the polyoxypropylene chains and weight percent ofoxyethylene groups.

The biologically-active copolymers of the present invention comprise asurface active compound with four hydrophobic segments and a smallproportion of hydrophile. Typical examples have eight segments oroctablock structure with a core of either a hydrophobic or hydrophiliccentral structure and a hydrophilic or hydrophobic outer structure asshown in the following schematic structures. ##STR3##

The entire molecule is poorly soluble in water and is either a nonionicor weakly cationic surface active agent. The steric configuration andphysiochemical properties of the molecule, rather than the chemicalnature of the constituent parts, are thought to be responsible for thebiologic effects of the copolymer.

The biologically active compounds of the present invention compriseblocks of polyoxypropylene and polyoxyethylene built on analkylenediamine initiator. The blocks of polyoxypropylene (POP) andpolyoxyethylene (POE) have the following structures: ##STR4##

The polymer blocks are formed by condensation of ethylene oxide andpropylene oxide at elevated temperature and pressure in the presence ofa basic catalyst. There is some statistical variation in the number ofmonomer units which combine to form a polymer chain in each copolymer.The molecular weights given are approximations of the average weight ofcopolymer molecule in each preparation. It is to be understood that theblocks of propylene oxide and ethylene oxide do not have to be pure.Small amounts of other materials can be admixed so long as the overallphysical chemical properties are not substantially changed. In addition,some double bonding may be present in the molecule due to eliminationreactions during synthesis of the molecule. All of these minorvariations in structure are not critical to the biological activity ofthe copolymer and are contemplated in the present invention.

A further description of the preparation of these block copolymers isfound in U.S. Pat. No. 2,674,619 and U.S. Pat. No. 2,979,528. (Also see"A Review of Block Polymer Surfactants", Schmolka, I. R., J. Am. OilChemists' Soc., 54: 110-116 (1977) and Block and Graft Copolymerization,Volume 2 edited by R. J. Ceresa, John Wiley & Sons, New York, (1976).

5.2 Chemical Structure

In one embodiment of the biologically active copolymers of the presentinvention, the block copolymer comprises a polymer of hydrophobicpolyoxypropylene (POP) built on an ethylenediamine initiator. Polymersof hydrophilic polyoxyethylene (POE) are then built on the block ofhydrophobic polyoxypropylene (POP). This results in an octablockcopolymer with the following general formula: ##STR5## wherein: the meanaggregate molecular weight of the hydrophobe portion of the octablockcopolymer consisting of polyoxypropylene (C₃ H₆ O)_(b) (POP) is betweenapproximately 5000 and 7000 daltons;

a is a number such that the hydrophile portion represented bypolyoxyethylene (C₂ H₄ O)_(a) (POE) constitutes between approximately10% to 40% of the total molecular weight of the compound;

b is a number such that the polyoxypropylene (C₃ H₆ O)_(b) (POP) portionof the total molecular weight of the octablock copolymer constitutesbetween approximately 60% and 90% of the compound; and

In another embodiment of the present invention, the block copolymercomprises a polymer of hydrophilic polyoxyethylene (POE) built on anethylene diamine initiator. Polymers of hydrophobic polyoxypropylene(POP) are then built on the block of hydrophilic polyethylene (POE).This results in an octablock copolymer with the following generalformula: ##STR6## wherein: the molecular weight of the hydrophobeportion of the octablock copolymer consisting of polyoxypropylene (C₃ H₆O)_(b) (POP) is between approximately 5000 and 7000 daltons;

a is a number such that the hydrophile portion represented bypolyoxyethylene (C₂ H₄ O)_(a) (POE) constitutes between approximately10% and 40% of the total molecular weight of the compound;

b is a number such that the polyoxypropylene (C₃ H₆ O)_(b) (POP) portionof the octablock copolymer constitutes between approximately 60% and 90%of the compound; and

this type of polymer is called reverse copolymer because its structureis the reverse of octablock copolymers that have polyoxypropylene (POP)in the center flanked by blocks of polyoxyethylene (POE).

The octablock copolymers comprising the biologically active copolymersof the present invention include, but are not limited to, the blockcopolymers Tetronic® and reverse Tetronic® manufactured by the BASFCorporation (BASF Corporation, Parsippany, N.J.).

A preferred biologically active copolymers is the octablock copolymerT130R2 (BASF Corporation, Parsippany, N.J.) which corresponds to thefollowing formula: ##STR7## wherein: the mean molecular weight of thehydrophobe portion of the octablock copolymer represented bypolyoxypropylene (C₃ H₆ O)_(b) (POP) is approximately 5750 daltons;

a is a number such that the hydrophile portion represented bypolyoxyethylene (C₂ H₄ O)_(a) (POE) constitutes approximately 20% of thecompound by weight; and

b is a number such that the polyoxypropylene (C₃ H₆ O)_(b) (POP) portionof the octablock copolymer constitutes approximately 80% of the compoundby weight.

Another preferred embodiment of the biologically active copolymers ofthe present invention is the compound designated T1501 (BASFCorporation, Parsippany, N.J.) which corresponds to the followingformula: ##STR8## wherein: the mean molecular weight of the hydrophobeportion of the octablock copolymer represented by polyoxypropylene (C₃H₆ O)_(b) (POP) is approximately 6750 daltons;

a is a number such that the hydrophile portion represented bypolyoxyethylene (C₂ H₄ O)_(a) (POE) constitutes approximately 10% of thecompound by weight; and

b is a number such that the polyoxypropylene (C₃ H₆ O)_(b) (POP) portionof the octablock copolymer constitutes approximately 90% of the compoundby weight.

The most preferred embodiment of the biologically active copolymers ofthe present invention is the octablock copolymer T150R1 (BASFCorporation, Parsippany, N.J.) which corresponds to the followingformula: ##STR9## wherein: the mean molecular weight of the hydrophobeportion of the octablock copolymer represented by polyoxypropylene (C₃H₆ O)_(b) (POP) is approximately 6750 daltons;

a is a number such that the hydrophile portion represented bypolyoxyethylene (C₂ H₄ O)_(a) (POE) constitutes approximately 10% of thecompound by weight; and

b is a number such that the polyoxypropylene (C₃ H₆ O)_(b) (POP) portionof the octablock copolymer constitutes approximately 90% of the compoundby weight.

5.3 Biological Activities

Although not wanting to be bound by the following theory, it is believedthat the biologically active polymers of the present invention act bythe following mechanism:

Investigators have demonstrated hormone-mediated interactions betweenthe thymus and the central nervous system. The biologically activecopolymers of the present invention are capable of affecting thisrelationship. It is believed that the present invention acts bymimicking the conformation and physicochemical properties of naturalhormones and neuropeptides. The biologic responses induced by thebiologically active copolymers of the present invention include:

1. Stimulation of rate and duration of growth of animals;

2. Gross morphologic changes in the uterus and adrenal glands;

3. Increased motor activity including excessive grooming;

4. Diuresis;

5. Noncytolytic release of histamine from mast cells by a temperature,calcium, energy dependent mechanism which is similar too from that ofsomatostatin and ACTH.

It is believed that there is a structural analogy between thebiologically active copolymers and hormones, such as somatostatin,adrenocorticotropic hormone (ACTH), and β-endorphin. These peptides havein common a sequence of basic amino acids adjacent to spans ofhydrophobic amino acids. These features are thought to be essential forfunction. Interaction of the oligoamine residues with cell surfaceanions and stabilization by adjacent hydrophobic moieties initiates themolecular program resulting in effector functions of the peptide. Theblock polymers have the same structural features: a central basicethylenediamine flanked by spans of hydrophobic polyoxypropylene (POP).By means of inhibition with monovalent amines, we have evidence that thecentral ethylenediamine groups function like the basic moieties ofpeptide hormones in stimulating release of histamine from mast cells.

In vitro studies provide direct evidence that the biologically activecopolymers of the present invention act in a manner analogous tosomatostatin and ACTH. Mast cells have been used as a model to studyreceptor mediated mechanisms through which these hormones induce theirbiological receptor mediated mechanisms. Minutes quantities of thepolymers cause histamine release from mast cells by atemperature-dependent process which requires energy and calcium and canbe blocked by specific inhibitors in a manner very similar to that ofsomatostatin and ACTH.

Both agonistic and antagonistic effects of hormones may be elicited bythe polymers. Biologic effects of the polymers vary with the structureof the polymer. The ability to modify the structure of these polymers tooptimize particular biologic effects provides potential to designsynthetic drugs with a precision and ease not possible in other systems.

Many chelating agents, such as ethylene-diaminetetraacetic acid (EDTA)consist of oligoamine sites flanked by hydrogen bonding groups. Theaddition of flanking hydrophobic moieties produces ionophores which areable to transport ions across lipid containing membranes. The compoundsof the present invention have this general structure and can act asionophores transporting cations across artificial membranes.Consequently, the compounds of the present invention represent a newchemical type of ionophore. Some neuropeptide hormones (e.g., substanceP) have ionophore activity.

The biologically-active copolymers of the present invention are alsoeffective in causing the cortex of the thymus to begin producing newT-lymphocytes thereby replenishing the immune system with these vitalregulatory cells. The copolymers also induce proliferation of largenumbers of post-thymic T-cells in the lymph nodes and other peripherallymphoid tissues. It is to be understood that stimulation of the immunesystem can lead to either an enhanced immune response or a suppressedimmune response depending upon which part of the immune system isstimulated. For example, if a population of suppressor t cells isstimulated, the suppressor t cells would suppress an immune response.

The biologically active copolymers of the present invention are alsoeffective in causing immunomodulation in poultry. It is believed thatthe copolymers induce precocious maturation of the bursa of Fabriciuswhich, in turn, enhances immune responsiveness.

The biologically-active copolymers of the present invention also exhibitadjuvant and inflammatory activities which vary depending on the lengthand arrangement of the blocks of polyoxyethylene and polyoxypropylene.The reverse octablock copolymers cause active, calcium dependenthistamine release from murine mast cells in vitro and the efficacy ofhistamine release is related to inflammatory activity in vivo.

The biologically active copolymers of the present invention wheninjected into an animal or a human with an antigen are effective inspecifically immunosuppressing the animal or human against that antigen.For purposes of definition, antigens can be broken down into two groups:immunogens and haptens.

Immunogens are compounds which, when introduced into a mammal, willresult in the formation of antibodies. Representative of the immunogensare proteins, glycoproteins and nucleoproteins, such as peptidehormones, serum proteins, complement proteins, coagulation factors, andviral or bacterial products. The following is a partial list ofrepresentative immunogens:

    ______________________________________                                        albumin             angiotensin                                               alpha-1-fetoprotein alpha-2-H globulin                                        bradykinin          calcitonin                                                carcinoembryonic antigen                                                                          chloriomamotropin                                         chorogonadotropin   corticotropin                                             coagulation factors microbiocidal products                                    erythropoietin      Factor VIII                                               fibrinogen          alpha-2-H globulin                                        follitropin         Gastrin                                                   fungal products     specific Immunogens                                       gastrin sulfate     glucagon                                                  gonadotropin        haptoglobin                                               Hepatitis B surface antigen                                                                       immunoglobulins                                           insulin             lipotropin                                                melanotropin        oxytocin                                                  myelin              myelin basic protein                                      nucleoproteins      peptide hormones                                          pancreozymin        placental lactogen                                        prathryin           proangiotensin                                            prolactin           somatotropin                                              proteins            glycoproteins                                             serum proteins      complement proteins                                       somatomadin         somatostatin                                              thryrotropin        vasotocin                                                 thymopoietin        vasopressin                                               viral products      bacterial products                                        ______________________________________                                    

Haptens are compounds which, when bound to an immunogenic carrier andintroduced into a chordate, will elicit formation of antibodies specificfor the hapten. Representative of the haptens are steroids such asestrogens and cortisones, low molecular weight peptides, other lowmolecular weight biological compounds, drugs such as antibiotics andchemotherapeutic compounds, industrial pollutants, flavoring agents,food additives, and food contaminants, and/or their metabolites orderivatives.

It is to be understood that the preferred biologically-active compoundmay differ in structure depending upon the biological activity that onedesires to elicit.

5.4 Administration of the Biologically Active Copolymers

The biologically-active copolymers of the present invention aregenerally poorly soluble in water. Although the compounds can beinjected into an animal or human in aqueous media, it is preferable thatthe biologically-active copolymers of the present invention be injectedas an oil-in-water or water-in-oil emulsion. A mineral oil or other oilysubstance such as Drakeol 6VR or Drakeol 5 (Penreco, Butler, Pa.) can beused as the oil phase of the emulsion. The aqueous phase can bephysiologic phosphate buffered saline or other physiologic saltsolution. The ratio of oil to water is preferably between approximately80:20 and 1:100.

Typically, an oil-in-water emulsion is prepared by mixing betweenapproximately 0.5 to 50 grams of the biologically active copolymers with5.0 ml of mineral oil in a Potter-Elvehjim homogenizer. Next, 95.0 ml ofphosphate buffered saline (0.85M sodium chloride, pH 7.3) containing0.2% polyoxyethylene sorbitan monooleate (Tween 80, Atlas ChemicalIndustries, Wilmington, Del.) and 50 mg bovine serum albumin (BSA, SigmaChemical Co., St. Louis, Mo.) is added. The mixture is homogenizedthoroughly to form a fine emulsion. The BSA and Tween 80 are used tostabilize the emulsion. It is to be understood that the method ofpreparing the emulsion, the proportions of oil and water and the type ofoil used are not critical. An effective emulsion could be prepared byusing a blender, by sonication or other means well known to those ofordinary skill in the art. It is to be further understood that othercarriers, emulsifiers, aqueous solutions and adjuvants that are known tothose of ordinary skill in the art can be used with the biologicallyactive copolymers of the present invention.

Water-in-oil emulsions are prepared as follows. A stock oil-emulsifiermixture is prepared by blending mineral oil with approximately 5% to 10%of a water-in-oil emulsifier. A mixture of 94.5% oil (Drakeol, Penreco,Butler, Pa.), 4.5% Sorbitan monooleate (Span 80, Atlas ChemicalIndustries, Wilmington, Del.) and 0.5% polyoxyethylene sorbitanmonooleate (Tween 80, Atlas Chemical Industries, Wilmington, Del.) iscommonly used. A commercial blend, Freund's incomplete adjuvant, (Difco,Detroit, Mich. or Sigma Chemical, St. Louis, Mo.) is also suitable.Approximately 0.5 to 5.0 grams of the biologically active copolymers isadded to either 60 ml of the oil-emulsifier mixture or to 40 ml of aphysiologic saline solution similar to that used in the oil-in-wateremulsion described above. The oil-emulsifier mixture is placed in ablender. The physiologic salt solution is added in three aliquots withvigorous homogenization to insure that a fine water-in-oil emulsion isprepared. Again, it is to be understood that the method of preparing theemulsion is not critical. Numerous variations of the composition of theaqueous and oil phases, their proportions and means of emulsificationwill be apparent to those skilled in the art and could be used withbiologically active copolymers in practicing the invention.

As an alternative, many of the nonionic block copolymers can besolubilized in a cold aqueous solution if first dissolved in a smallvolume of ethanol.

The biologically active copolymers of the present invention areeffective with only one injection of compound being administered to ananimal. However, in certain cases, subsequent injections may benecessary to achieve maximum stimulation of the immune system or otherdesired effect. The mode of injection can be subcutaneous, intramuscularor intravenous. A preferred mode of injection is subcutaneous.Intravenous injection is hazardous because of the toxic effects ofembolic emulsions.

The optimum amount of the biologically active polymers in an injectionvaries with the size of the animal being treated. With animals such asrats or mice, the optimum amount of biologically active copolymers isbetween approximately 0.5 and 5 mg per animal. With larger animals alarger quantity of biologically active copolymers is required in theinjection for optimum results. With humans, cattle or swine, the dosevaries with the age, size and condition of the individual butapproximates 5 to 500 mg in most cases.

It has been determined that the octablock copolymers can be administeredorally and have an effect on the growth of animals. Thus, it iscontemplated in the present invention that the biologically-activecopolymers can be administered orally, either in water or as an additiveto food.

The following specific examples will illustrate the invention as itapplies in particular to stimulating the immune system in mice and rats.It will be appreciated that other examples will be apparent to those ofordinary skill in the art and that the invention is not limited to thesespecific illustrative examples.

6.0 Brief Description of the Figures

FIG. 1 shows histamine release from mast cells that have been exposed toseveral copolymers of the present invention.

FIG. 2 shows influx of sodium ions into red blood cells that have beenexposed to several copolymers of the present invention.

FIG. 3 shows the relationship between footpad swelling and sodium fluxand several of the copolymers of the present invention.

FIG. 4 shows the effect of T150R1 on mouse thymus in vivo.

FIG. 5 shows the effect of the copolymer of the present invention onmotor activity in rats.

FIG. 6 shows the differential tumoricidal activity of T150R1 on K-652cells and HL-60 cells as measured by cell viability.

FIG. 7 shows the differential tumoricidal activity of T150R1 on K-652cells and HL-60 cells as measured by tritiated thymidine uptake.

FIG. 8 shows the differential tumoricidal activity of T150R1 and T150R2on K-652 cells and HL-60 cells as measured by release of lacticdehydrogenase.

FIG. 9 shows active immunosuppression by T150R1.

FIG. 10 shows adoptive immunosuppression by cells from a rat treatedwith T150R1.

FIG. 11 shows stimulation of thymus cell proliferation by T150R1 inLewis rats as measured by increased number of cells.

FIG. 12 shows stimulation of thymus cell proliferation by T150R1 inLewis rats as measured by increased in vivo incorporation of tritiatedthymidine.

7.0 EXAMPLES 7.1 Mast Cell Degranulation

The mast cell degranulation activity of several of the copolymers of thepresent invention are shown in this Example. Block copolymers wereobtained from BASF Corporation, Parsippany, N.J., and are shown in thefollowing Table A.

                  TABLE A                                                         ______________________________________                                                       Mean.sup.a, b                                                  Copolymer      Mol. Wt..sup.a                                                                          Structure                                            ______________________________________                                        T130R1         6,800     N- -4-26                                             T130R2         7,740     N- --10-26                                           T150R1         8,000     N- -5-32                                             T150R4         11,810    N- --27-32                                           T150R8         20,400    N- --92-32                                           ______________________________________                                         .sup.a Data from Manufacturer                                                 .sup.b Amine nitrogen followed by mean number of ethoxy units (underlined     followed by mean number of propoxy units per linear chain                

The copolymers in Table A range in physical form from viscous liquids toflakes. Measurement of the liquid copolymers was made using a positivedisplacement pipet based on a density approximately equal to that ofwater (range 1.01-1.03). Since the solubility of these materials inaqueous medium decreases with increasing temperature, all dilutions weremade in cold (4° C.) buffer.

Mouse peritoneal mast cells were obtained by lavage from female C3H mice(Charles River, Wilmington, Mass.). For each experiment one or twoanimals were sacrificed using dry ice vapors and the peritoneal cavityinjected with 10 ml ice cold Dulbecco's phosphate-buffered saline(hereinafter PBS) (Gibco, Grand Island, N.Y.) containing 10 U/mlheparin. The peritoneum was massaged for 30 seconds and the fluidwithdrawn. Cells were washed twice in PBS and counted using Alcian blue.The average yield was 4-6×10⁶ cells/animal with mast cells comprising3-5% of the total cells. Cells were resuspended in Tyrode's solution(see Pearce, F. L. and White, J. R. (1984) Agents Actions 14, 392)containing 1 mg/ml bovine serum albumin (Miles Laboratories, Naperville,Ill.). Tyrode's solution was made using distilled, deionized water with137 mM NaCl, LiCl, or KCl as the major cation species with 1 mM CaCl₂added as indicated.

Cells were prewarmed to 37° C. for 5 minutes and then added in 200 μlaliquots (2-5×10³ mast cells) to polypropylene tubes containing an equalvolume of buffer with or without copolymer. Total histamine content ofthe cells was obtained by lysing the cells in 3% perchloric acid. Cellswere incubated for 30 minutes at 37° C. and then centrifuged for 5minutes at 1500×g at 4° C. Supernatants were analyzed for histaminecontent using the automated fluorometric method (See Siraganian, R. P.(1974) Anal. Biochem. 57, 383). All samples were performed in duplicateand the results are presented as means of sample pairs. Net percenthistamine release was calculated by the following formula: ##EQU1##

All of the copolymers shown in Table A were tested for their ability tocause non-cytolytic histamine release. The five copolymers wereincubated at a concentration of 100 μg/ml with the mast cells. At theend of 30 minutes incubation with the copolymer, the percent nethistamine release was measured. These results are summarized in FIG. 1.As shown, the rates obtained for these copolymers revealed a spectrum ofactivity with the T130R2 copolymer showing the greatest histaminerelease and the T150R8 showing the least activity.

7.2 Ionophore Activity

The ionophore activity of the biologically active copolymers wasdetermined by measuring the influx of ions into human red blood cells.

Human blood anticoagulated with lithium heparin was washed 3 times in0.9% NaCl by centrifugation for 10 minutes at room temperature at 150×gand diluted to 10% hematocrit in saline. For Na⁺ flux measurements thesaline contained approximately 5 μCi ²² Na⁺ /ml (Amersham Corp.,Arlington Heights, Ill.). For determination of Ca⁺⁺ flux, cells werediluted in saline containing 4 mM CaCl² with ⁴⁵ Ca⁺⁺ tracer (ICNBiomedicals, Inc., Irvine, Calif.) at approximately 10 μCi/ml. Cellswere warmed to 37° C. in a water bath and then added to equal volumes ofpre-warmed buffer containing copolymers and mixed by vortexing. For Na⁺or Ca⁺⁺ flux measurements, 50 μl duplicate aliquots of each wholesuspension were removed during incubation to measure total activity. Atselected time points, 200 μl duplicate aliquots were removed and addedto 1.5 ml minicentrifuge tubes (American Scientific Products, McGawPark, Ill.) containing 200 μl SF1154 silicone oil (General Electric Co.,Waterford, N.Y.).

For Na⁺ and Ca⁺⁺ measurements, the supernatants above the oil wereaspirated and the portion of the tube above the oil was washed twice bycarefully adding and then aspirating distilled water. After removal ofmost of the oil the cells were resuspended in 250 μl distilled water.Residual ²² Na⁺ activity was measured using a gamma counter andremaining ⁴⁵ Ca⁺⁺ activity was measured in a beta counter after mixing100 μl of the lysate with 3 ml Opti-Fluor LSC cocktail (UnitedTechnologies Packard). Determination of potassium efflux was performedas above except that the cells were mixed with copolymer in normalsaline at a 10% final hematocrit. After centrifugation, supernatantswere removed and assayed for potassium content by flame emissionspectroscopy using an IL 443 Flame Photometer (InstrumentationLaboratory, Inc., Lexington, Mass.).

Residual counts in the pellets or the amount of K⁺ in the supernatantswere normalized for pellet hemoglobin content which was determined usingDrabkin's solution. There was no difference between hemoglobin contentof pellets incubated with any of the copolymers used or with bufferalone, indicating that copolymers are not cytolytic. All experimentswere preformed at least in duplicate.

Five copolymers were tested for their ability to cause influx of Na⁺ions over a period of 30 minutes. The results of these tests aresummarized in FIG. 2.

Comparison of the rates obtained for these copolymers revealed aspectrum of activity which correlated with their ability to trigger invitro histamine release from murine mast cells and in vivo inflammationas determined by peak footpad swelling following subplantar injection inmice. These results are summarized in FIG. 3.

The larger the sodium flux observed with each copolymer, the morehistamine release and inflammation the copolymer induced.

7.3 Histamine Release vs. Footpad Swelling

Three octablock and seven reverse octablock copolymers were obtainedfrom the BASF, Parsippany, N.J. Resident peritoneal leukocytes wereobtained by lavage as described in Example 7.2.

Six of seven reverse octablock copolymers were found to releasesignificant amounts of histamine from mouse peritoneal mast cells. Theseresults are summarized in Table B.

                                      TABLE B                                     __________________________________________________________________________    Histamine Release Compared to                                                 Footpad Swelling Induced by Copolymers                                        Avg.sup.a  Chain.sup.b                                                                        % Histamine Released.sup.c                                                                Peak Footpad.sup.d                                Copolymer                                                                           MW   Structure                                                                          10 μg/ml                                                                         100 μg/ml                                                                        Swelling (mm)                                     __________________________________________________________________________    T130R2                                                                               7,740                                                                             N- --10-26                                                                         58.0 ± 8.6                                                                       66.3 ± 6.5                                                                       2.92 ± 0.41                                    T130R1                                                                               6,800                                                                             N- -4-26                                                                           16.3 ± 4.0                                                                       29.0 ± 7.0                                                                       1.65 ± 0.3                                     T110R1                                                                               5,220                                                                             N- -3-21                                                                           16.7 ± 2.6                                                                       15.7 ± 2.6                                                                        1.3 ± 0.33                                    T90R1  4,580                                                                             N- -3-18                                                                           15.7 ± 2.6                                                                       13.3 ± 2.5                                                                       1.18 ± 0.24                                    T150R1                                                                               8,000                                                                             N- -5-32                                                                            9.0 ± 0.8                                                                       11.7 ± 3.1                                                                       1.17 ± 0.08                                    T150R4                                                                              11,810                                                                             N- --27-32                                                                          0.3 ± 0.5                                                                       16.3 ± 7.9                                                                       1.04 ± 0.5                                     T150R8                                                                              20,400                                                                             N- --92-32                                                                          0.7 ± 0.5                                                                        0.0 ± 0.0                                                                       0.79 ± 0.01                                    __________________________________________________________________________     .sup.a Average Molecular Weight (data from manufacturer)                      .sup.b Reverse octablocks are 4chain structures arranged around a core of     ethylenediamine moiety. Chain structure is here represented by the amine      nitrogen (N) followed by the average number of POE blocks (underlined) an     the number of POP blocks in one chain.                                        .sup.c Mean histamine content of supernatants from cells incubated for 30     minutes at 37° C. with copolymers at the indicated concentrations.     Mean ± standard deviation for three experiments. Values are corrected      for spontaneous release which was always less than 10%.                       .sup.d Rank order of peak footpad swelling produced by subplantar             injection of 50 μl of an oil in water emulsion containing 1.25 mg          copolymer (mean ± SE on groups of 10 mice.)                           

The amount of histamine released by the various copolymers was relatedto the level of inflammation at the site of injection, i.e., the moreinflammatory the copolymer in vivo, the more histamine release in vitro.

7.4 Thymus Stimulation Activity

Six week old female ICR outbred mice were injected in the rear footpadswith an oil-in-water emulsion containing copolymer 1.25 mg T105R1, 2.5mg mineral oil and 25 μg bovine serum albumin in 0.05 ml of 0.01Mphosphate buffered saline containing 0.1% Tween 80. Identical injectionswere made into both rear footpads. Controls were animals that wereinjected with emulsions without the T150R1. Animals were sacrificed at 4days, 1, 2, 3 and 6 weeks. Their thymuses were carefully dissected freeof connective tissue and were weighed.

Microscopic examination revealed that the changes in the thymus werelimited almost exclusively to the cortical lymphocytes. Their numberswere markedly reduced three days after injection, but approached normalat one week. After the transient decrease in the number of lymphocytes,the size of the thymus glands of treated animals increased markedly overthose of controls. The medullary areas demonstrated little change, butthe cortical areas were much larger than normal. The cortical areas ofthe thymus were composed of immature and mature small lymphoid cells.The reduction in size of the thymus in controls was the expected resultof normal involution with aging. Thus, the injection of T150R1 preventedinvolution during the time studied. In several experiments carried outwith identical protocol, the thymuses of treated animals wasconsistently nearly twice as large as those of the controls. Sometreated mice were followed for over eighteen months and appear healthy.

Table C shows that the inhibition of involution of the thymus is longterm. The weights of the body, thymus and spleens were after 18 months.

                  TABLE C                                                         ______________________________________                                                        injected at  injected at                                      Control         4 weeks      6 weeks                                          (n = 3)         (n = 3)      (n = 3)                                          ______________________________________                                        body    40.7 ± 7 g                                                                              47.0 ± 4.2 g                                                                            53.3 ± 1.6 g                             thymus  34.7 ± 5.0 mg                                                                           67.6 ± 18.7 mg                                                                          66.2 ± 26 mg                             spleen 126.7 ± 47.3 mg                                                                         180.7 ± 61.0 mg                                                                         122.7 ± 19 mg                             ______________________________________                                    

Histological examination of the thymuses showed little thymus tissue incontrol mice. Thymuses of injected mice had "young looking" thymustissue.

7.5 Thymus Stimulation, Copolymer in Saline

Mice were injected via the tail vein with the following preparations:

1. Saline at 4° C.

2. 2.5 mg of copolymer T150R1 in saline at 4° C.

3. 1μ beads coated with T150R1 copolymer.

Each experimental group contained four mice. After 6 days, the mice weresacrificed and the thymuses were carefully dissected and weighed. Theresults of this experiment are summarized in FIG. 4.

This Example shows that the copolymer dissolved in cold saline andinjected directly into the blood of an animal causes a marked increasein the weight of the thymus.

7.6 Thymus Stimulation, Oil and Water Emulsion

Mice were injected in the footpad with related copolymers and otherforms of emulsion. Copolymers T130R2 and T1501 had weaker effects instimulating the increased size of the thymus. The results of anexperiment with water in oil emulsions is shown in Table D.

                  TABLE D                                                         ______________________________________                                                    WATER-IN-OIL EMULSIONS                                                        Weight of the thymus in mg                                        Copolymer     2 weeks     6 weeks                                             ______________________________________                                        T150R1        65 ± 7   110 ± 7                                          T1501          84 ± 15 77 ± 4                                           Vehicle Control                                                                             101 ± 18 50 ± 7                                           ______________________________________                                    

7.7 Thymus Stimulation, General Observations

Mice were treated as in Example 7.6 and carefully observed for a periodof several months. The following observations were made. The treatedmice were larger than control mice. The treated mice appeared to exhibitincreased motor activity including more general activity in the cage,excessive grooming and the treated mice ate more than the control mice.

The uterus of the treated female mice was larger and appeared to haveincreased vascularity. There was an increased prominence of Peyer'spatches (gut associated lymphoid tissue) in the treated animals.

7.8 Growth Stimulation, Comparison of Copolymers

The biologically active copolymer of the present invention wasadministered to mice. The growth of the mice was then measured after sixweeks.

Oil-in-water emulsions were prepared with 1 mg bovine serum albumin(BSA), 50 mg of the indicated copolymer (all copolymers weremanufactured by BASF Corporation, Parsippany, N.J.) and 100 μl mineraloil (Drakeol 6VR, Penreco Refining Company, Butler, Pa.) in 2 ml ofphosphate-buffered saline (PBS) with 0.2% Tween 80 (Sigma Chemical Co.,St. Louis, Mo.). The mixture was homogenized in a Potter Elvehjimhomogenizer. The oil, copolymer and BSA were homogenized together beforeadding the phosphate buffered saline. The mixture was then furtherhomogenized to form a fine emulsion.

Emulsions were prepared of each of the copolymers shown in Table D. Eachof these copolymers is comprised of blocks of polyoxyethylene (POE) andpolyoxypropylene (POP) arranged in the fashion previously described forcopolymer T150R1. They differ from one another only in the size of theblocks. Each has characteristic physiochemical and biologic properties.

Mice were injected with 50 μg of BSA, 2.5 mg copolymer and 5 mg oil in0.1 ml of an oil-in-water emulsion divided between the two hind feet.Their weights six weeks later are summarized in Table E.

                  TABLE E                                                         ______________________________________                                                Molecular Wt Percent                                                  Copolymer                                                                             (Hydrophobe) (Hydrophile).sup.a                                                                       Mean Wt.sup.b                                 ______________________________________                                        T90R1   3750         10%        22.7 ± 1.03 gm                             T110R1  4750         10%        22.7 ± 0.76 gm                             T130R1  5750         10%        23.3 ± 1.36 gm                             T130R2  5750         20%        24.7 ± 0.62 gm                             T150R1  6750         10%        26.7 ± 1.75 gm                             T150R4  6750         40%        23.5 ± 1.77 gm                             T150R8  6750         80%        23.0 ± 1.06 gm                             ______________________________________                                         .sup.a Percentages are to the nearest 10%                                     .sup.b Mean weight of groups of five mice ± standard deviation        

The mice injected with the emulsion containing the copolymer T150R1 werevisibly larger than those injected with any of the other copolymers. Allof the mice appeared healthy.

All of the copolymers used in Table E consist of blocks ofpolyoxypropylene (POP) and polyoxyethylene (POE) attached in the samereverse octablock configuration. The compounds differ from one anotheronly in the size of the blocks of each compound. These differences,however, confer distinct physicochemical properties on the copolymerswhich correlate with their biologic activities.

7.9 Growth Stimulation, Effect of Various Emulsions

Oil-in-water emulsions were prepared using 1 mg bovine serum albumin(BSA), 50 mg of either copolymer T1501 or T150R1 (BASF Corporation,Parsippany, N.J.) and 100 μl mineral oil (Drakeol 6VR, Penreco, Butler,Pa.) in 2.0 ml phosphate buffered saline with 0.2% Tween 80 in a PotterEvehjin homogenizer as described above. The control oil-in-wateremulsion was prepared identically except that no copolymer was used.

Copolymer T1501 has the following general structure: ##STR10## andcopolymer T150R1 has the following general structure: ##STR11##

The hydrophobic polyoxypropylene block in each of the structures have amean aggregate molecular weight of approximately 6750. They each containapproximately 10% hydrophilic polyoxyethylene.

The water-in-oil emulsion was prepared using 50 mg of either copolymerT1501 or T150R1, 1.2 ml Freund's incomplete adjuvant (Sigma ChemicalCompany, St. Louis, Mo.), and 0.8 ml Hank's balanced salt solution,(Gibco, Grand Island, N.Y.), containing 1 mg bovine serum albumin (BSA).The control emulsion was prepared identically except that it containedno copolymer.

Mice were injected with 2.5 mg of copolymer in 0.1 ml of either theoil-in-water or water-in-oil emulsion. Controls consisted of miceinjected with the same dose of an identical emulsion which contained nocopolymer. The injections were given subcutaneously and the total dosewas divided between the two hind feet. Their weights at six weeks aresummarized in Table F.

                  TABLE F                                                         ______________________________________                                                   MEAN WEIGHT.sup.a                                                               Oil-in-water Water-in-oil                                        COPOLYMER    Emulsion     Emulsion                                            ______________________________________                                        T1501        21.2 ± 1.92 grams                                                                       26.6 ± 1.18 grams                                T150R1       28.1 ± 0.72 grams                                                                       26.5 ± 2.32 grams                                Control      22.5 ± 0.96 grams                                                                       23.0 ± 1.41 grams                                ______________________________________                                         .sup.a Mean weight of groups of five mice ± standard deviation        

The block copolymer designated T150R1 was highly effective instimulating growth in both water-in-oil emulsion and in oil-in-wateremulsion. The block copolymer designated T1501 was effective instimulating growth in a water-in-oil emulsion.

7.10 Stimulation of Motor Activity

The effect the biologically-active copolymer of the present inventionhas on the motor activity of rats was measured. The animals used in thisExample were adult male Sprague-Dawley rats.

The rats were individually housed in suspended cages. All rats werehoused for three weeks in the same room prior to initiation of thestudy.

Baseline measurements of horizontal locomotor activity were made for oneweek prior to administration of the copolymer. Horizontal activity wasmeasured using an activity box (Omnitech, Inc., Columbus, Ohio)interfaced with a Vic 20 microcomputer. The activity box has two rows of8 photocells positioned at 90° angles. When the light path to aphotocell is broken, a voltage drop occurs and is recorded by thecomputer. Activity determinations were made on all nine rats at threeday intervals.

The test and control solutions were prepared as follows: The aqueousphase was prepared by mixing 100 ml of PBS and 0.2 ml of Tween-80. Thismixture was stirred approximately 5 minutes. 200 μl of oil (Drakeol,Penreco, Butler, Pa.) was added to 100 μl of T150R1. This mixture ofbiologically active copolymer and oil was homogenized for two minutes ina Potter-Elvinjim homogenizer. Next, 1 ml of PBS with 2% Tween-80 wasadded and homogenized for another 2 minutes. Finally, an additional 1 mlof PBS with 2% Tween-80 was added and homogenized for 2 minutes. Thecontrol emulsion was prepared as above but without the addition ofT150R1 copolymer.

All rats were injected with 100 μl of either the control or the testemulsion. The test emulsion contained a total of 5 mg of T150R1copolymer. All injections were given subcutaneously on the dorsum of theneck. The effect of the compound on the activity of rats is shown inFIG. 5.

As shown in FIG. 5, rats that were injected with the biologically activecopolymer of the present invention demonstrated markedly increasedactivity over the control.

7.11 Tumoricidal Activity

The effects of the biologically-active copolymer of the presentinvention on a human leukemic cell line were evaluated. Thebiologically-active copolymer (T150R1) tested had the following formula:##STR12## wherein: the mean molecular weight of the hydrophobe portionof the octablock copolymer represented by polyoxypropylene (C₃ H₆ O)_(b)(POP) is approximately 6750 daltons;

a is a number such that the hydrophile portion represented bypolyoxyethylene (C₂ H₄ O)_(a) (POE) constitutes approximately 10% of thecompound by weight; and

b is a number such that the polyoxypropylene (C₃ H₆ O)_(b) (POP) portionof the octablock copolymer constitutes approximately 90% of the compoundby weight.

This biologically active copolymer is designated T150R1.

The effects of the biologically active copolymer T150R1 of the presentinvention on malignant human cells were evaluated by incubating variousdoses of the copolymer with K562 acute myeloblastic leukemia cells orHL-60 promyelocytic leukemia cells and determining viable cell numbersby the trypan-blue exclusion method and cellular proliferation asmeasured by incorporation of ³ H-thymidine into DNA after two days ofculture in CEM medium containing 5% fetal calf serum. The result areshown in FIG. 6 and 7.

FIG. 6 shows that the biologically-active copolymer of the presentinvention was highly toxic to HL-60 cells, but only minimally affectedthe viability of K-562 cells. With a dose of biologically-activecopolymer of 10 μg per 10⁵ cells, the viability of HL-60 cells decreasedto less than 35% that of control cells incubated with medium only. With30 μg/10⁵ cells of biologically-active copolymer, over 90% of the cellswere killed. In contrast, over 80% of the K-562 cells were alive withthe 10 μg dose and over 70% of the K-562 cells were alive with the 30 μgdose of biologically-active copolymer.

As shown in FIG. 7, the biologically-active copolymer of the presentinvention strikingly inhibited DNA synthesis by both HL-60 and K562. TheHL-60 was more greatly affected than the K562 cell line. However, withthe 30 μg dose at which greater than 70% of the K562 cells in theculture were alive, DNA synthesis by these cells was inhibited greaterthan 85%.

Thus, the biologically-active copolymer of the present invention showedboth cytotoxic and cytostatic effects against two human leukemia celllines. The fact that HL-60 was much more susceptible than was K562 showsthat the copolymer was selective for the HL-60.

7.12 Differential Effect of Copolymers on Leukemic Cell Lines

FIG. 8 shows the results of differential effect of the biologicallyactive copolymers of the present invention on two different cell lines.In addition to the biologically-active copolymer T150R1 tested inExample 7.11, T130R1 represented by the following formula: ##STR13##wherein: the mean molecular weight of the hydrophobe portion of theoctablock copolymer represented by polyoxypropylene (C₃ H₆ O)_(b) (POP)is approximately 5750 daltons;

a is a number such that the hydrophile portion represented bypolyoxyethylene (C₂ H₄ O)_(a) (POE) constitutes approximately 10% of thecompound by weight; and

b is a number such that the polyoxypropylene (C₃ H₆ O)_(b) (POP) portionof the octablock copolymer constitutes approximately 90% of the compoundby weight.

Cell cultures were incubated with various concentrations of the twocopolymers. After 24 hours of incubation, the level of lacticdehydrogenase (LDH) was measured in the supernatant and compared to thetotal LDH in untreated cells. The release of LDH into the surroundingmedium is a measure of cell death. As shown in FIG. 8, the two celllines responded very differently to the two drugs.

Thus, the tumoricidal compound of the present invention showed bothcytotoxic effects against two human leukemia cell lines. HL-60 cell linewas more sensitive to the T150R1 while the K-562 was much more sensitiveto the T150R2 copolymer.

7.13 Differentiation of Leukemic Cell Lines

The effects of low concentrations of octablock copolymer T130R2 weredetermined on the HL-60 cell lines. T130R2 octablock copolymer causesinhibition of the proliferation and retards DNA synthesis over time.T130R2 causes cell lysis at concentrations greater than 10 μg/ml after a24 hour incubation. However, at lower concentration, T130R2 causes adose dependent increase in cell adherence starting at a concentration ofapproximately 1 μg/ml to a concentration of approximately 30 μg/ml.After 24 hours of incubation, over 50% of the normally non-adherentHL-60 cells are adherent. The data indicates that the HL-60 cellsdifferentiate when exposed to the T130R2 copolymer. The evidence thatsupports this observation is as follows: (a) An incubation of at least 2days was required to arrest growth; (b) T130R2-treatment caused cells toadher to the bottom of the plate, which is an indication ofdifferentiation into monocytic cells; (c) cell-cycle analysis showed twolevels of blockade, namely, G0+G1-S and G2+M-G0+G1 phases of cell-cycle.

7.14 Immunosuppressive Activity

In an experiment to investigate the effects of T150R1 on antibodyproduction, groups of four mice were administered the antigen, bovineserum albumin, in an oil-in-water emulsion with or without T150R1. Themice were challenged 14 days later with BSA plus an adjuvant and theantibody response to the antigen was measured at 1, 2, and 3 weeks. Asshown in FIG. 9, antibody titers in the T150R1 treated animals were lessthan 1000:1 at all three time points whereas the control mice displayedincreasing titers with an average titer greater than 4000:1 at threeweeks following challenge.

7.15 Adoptive Transfer of Immunosuppression

In another experiment, mice were administered BSA in an oil-in-wateremulsion with or without T150R1 as in the above experiment. Mice weresacrificed 14 days later. Thymus and spleen were removed and single cellsuspensions of thymus or spleen were administered intravenously tosyngenic recipient mice. Mice were challenged at the same time with BSAin an oil-in-water emulsion with adjuvant. Titers to the BSA weremeasured at 1, 2, and 3 weeks. These results are shown in FIG. 10.

As shown in FIG. 10, mice receiving thymus cells had titers below 1000:1at all three time points. Those receiving spleen cells had titers below1000:1 at 1 and 2 weeks and below 3500:1 at 3 weeks. Control mice whichdid not receive thymus or spleen cells had steadily increasing titerswhich at three weeks averaged greater than 4000:1. This experimentdemonstrates adoptive transfer of immunosuppression with spleen andthymus cells.

7.16 Suppression of Experimental Allergic Encephalomyelitis

The effect of T150R1 on the progression of experimental allergicencephalomyelitis (EAE), a well characterized animal model of multiplesclerosis, has been studied. In this animal model EAE is induced byinjecting myelin basic protein (MBP), a component of the sheath coveringcertain nerves, in combination with complete Freund's adjuvant (CFA), apotent adjuvant, into Lewis rats, a strain particularly prone todeveloping autoimmune disease. Over a two week period followinginjection, the rats develop an ascending flaccid paralysis andcharacteristic lesions of the central nervous system. In a studydesigned to investigate T150R1, rats were injected with MBP-CFA with orwithout T150R1. Animals receiving MBP-CFA developed signs typical ofEAE. The T150R1 rats however, showed no evidence of paralysis. All ratswere sacrificed at day 13 following injection, the thymuses were removedand single cell suspensions were made and cells were counted. As shownin FIG. 11, the thymus of T150R1 treated rats contained over 40 timesthe number of cells as the MBP-CFA only treated rats. As an additionalcontrol, the response to hen egg albumin injected with CFA and T150R1was evaluated. Theses rats also had a greatly increased number of cellsin the thymus.

The proliferative activity of thymus cells from T150R1 treated rats, asmeasured by tritiated thymidine uptake, was also markedly increased overthe MBP-CFA animals. The in vitro response to challenge with MBP wasalso evaluated by measuring tritiated thymidine uptake. In thisexperiment, T150R1-MBP-CFA treated animals demonstrated higher levels ofactivity than the MBP-CFA treated animals at 24 hrs (See FIG. 12).However, at 72 and 96 hours T150R1 treated animals had negligiblethymidine incorporation while the MBP-CFA rats maintained levels ofincorporation comparable to the 24 hour value.

Although not wanting to be bound by the following theory, it is believedthe increase cellularity in the thymus is due to thymic stimulation withthe dominant response being stimulation of specific suppressor cells.The ability to induce a specific suppressor cell response hasimplications for those diseases or conditions in which a specifictolerant state is desired.

7.17 Immunomodulation in Poultry, Bursal Weight

Chickens are treated subcutaneously at one day of age with anoil-in-water emulsion of the copolymer T150R1. A 2.5 mg dose of thecopolymer is administered. Relative bursal weights at 1, 3, and 6 weeksof age are determined and the results are set forth in Table G. Acomparison is made of chickens treated with the polymer in theoil-in-water emulsion against chickens treated with the oil-in-watervehicle only. No difference in bursal weight is found at 1 week of age.Male and female chickens treated with the polymer have significantlysmaller bursae at 3 weeks of age than chickens injected with the vehiclealone. At 6 weeks of age, there is no difference in bursal weights ofthe treated and untreated female chickens, but the difference in malesis more pronounced. In fact, many of the male birds have effete bursaeat 6 weeks. It is known in the art that testosterone and its analoguescause bursal atrophy which probably is due to enhanced B cellemigration. The pronounced effect on bursal weights in males in thisexperiment may be due to an additive effect of testosterone whichreaches significant levels around the fourth week of age.

Antibody assays are done with sera collected from the treated chickensby commercial ELISA tests. It can be concluded from these tests thatbursal atrophy is not caused by adventitial infection with infectiousbursal disease virus as representative serum samples do not havemeasurable levels of antibodies to it, or to other common viruses ofpoultry. The observed results therefore are most likely the result oftreatment with T150R1.

                                      TABLE G                                     __________________________________________________________________________    Relative bursal weights.sup.a                                                           Females   Males     Unsexed                                         Age (wks) T150R1                                                                        Vehicle                                                                            T150R1                                                                             Vehicle                                                                            T150R1                                                                             Vehicle                                         __________________________________________________________________________    1         --   --   --   --   0.174                                                                              0.173                                      3         0.314                                                                              0.292.sup.b                                                                        0.324                                                                              0.239.sup.c                                                                        --   --                                         6         0.243                                                                              0.247.sup.                                                                         0.263                                                                              0.194.sup.c                                          __________________________________________________________________________     .sup.a Mean relative bursal weights of 125 chickens per group at 1 week,      and 225 chickens per group at 3 and 6 weeks                                   .sup.b Significantly different from vehicle control, p = 0.0014, pooled t     test.                                                                         .sup.c Significantly different from vehicle control, p = 0.0000, pooled t     test.                                                                    

7.18 Immunomodulation in Poultry, Bursal Morphology

Chickens are treated subcutaneously at one day of age with anoil-in-water emulsion of the copolymer T150R1. The doses administeredcontain 1.0, 2.5 and 5.0 mg of the copolymer.

Morphometric data concerning the bursae of the treated birds arecollected by using a SONY® television camera mounted on a lightmicroscope to project images of tissue sections of bursae on a smoothsurface. An electronic stylus is used to circumscribe bursal folliclesalong their outer perimeters and to trace the outer and inner(medullary) margins of the interfollicular epithelium (IFE). The stylusis coupled to an Apple IIe computer which employs a program to convertthe electronic signals to square inches of area inscribed. A metric gridis projected and measured with the stylus in order to convert squareinches to μm². The cortical area is defined as the difference betweenfollicular area and the area inscribed by the outer margin of theinterfollicular epithelium. The area of the interfollicular epitheliumis defined as the difference between the medullary area and the areainscribed by the outer margin of the interfollicular epithelium.Twenty-five contiguous lymphoid follicles are measured in each bursa andthe coefficient of variance (CV) of these measurements range from10-35%. Each group analyzed has 20 chickens. The CV for the 20observations ranges from 5-15%. The data collected at three days, oneweek, two weeks and three weeks after treatment are presented in TablesH-K.

At three days of age, the medullae of chicks treated with 1.0 and 2.5 mgT150R1 are larger than medullae of the controls. This enlargement isalso reflected in the cortico-medullary (C/M) ratios. Although themedullae tend to be smaller and the cortices larger in treated birdsthan in controls at 1 week of age, these trends are not statisticallysignificant until they are considered together in the cortico-medullaryratios. Because the follicles of chickens treated with 1.0 mg T150R1 areunusually small at two weeks (as shown in Table J), a direct comparisonwith the other dosage groups is risky. Nevertheless, at two weeks thecortico-medullary ratio is higher than controls and not different frombirds treated with 2.5 mg of the copolymer. At three weeks, thecortico-medullary ratio of birds treated with 2.5 mg T150R1 are greaterthan controls, but the difference is not as great and the previousperturbation in bursal development probably is subsiding. Although a fewdifferences are observed in areas of the interfollicular epithelium, nopattern can be discerned and these differences are consideredinconsequential. As shown in Tables I, J, and K, the 5.0 mg dose appearsto be less effective than the 2.5 mg dose. Apparently, a dose containing1.0 mg or 2.5 mg of the copolymer T150R1 accelerates the maturation ofthe bursa of Fabricius.

Accordingly, precocious maturation of bursae should lead to more rapidgrowth of medullae which should begin to regress early. Early migrationof medullary cells to the cortices should result in more rapid expansionof cortices than normal. As the cortical lymphocytes mature and migrateto the spleen, bursal involution should occur at an earlier age, and beevident in males earlier than females. These are the changes observed.

                  TABLE H                                                         ______________________________________                                        Morphometry of Lymphoid Follicles                                             Three Days after Treatment.sup.1, 2                                           Treatment                                                                              Follicle Medulla  IFE    Cortex C/M                                  ______________________________________                                        None     28,900   3,340.sup.a                                                                            7,280.sup.a                                                                          18,300 5.6.sup.a                            Vehicle  28,800   3,340.sup.a                                                                            8,360.sup.a, b                                                                       17,000 5.0.sup.a, c                         1.0 mg   33,400   6,050.sup.b                                                                            9,670.sup.b                                                                          17,700 2.9.sup.b                            2.5 mg   31,800   4,580.sup.c                                                                            9,130.sup.a, b                                                                       18,000 4.1.sup.c                            5.0 mg   26,400   3,000.sup.a                                                                            7,460.sup.a, b                                                                       16,000 5.4.sup.a                            ______________________________________                                         .sup.1 Values are mean areas of 25 contiguous follicles, their medullae,      cortices and zones of interfollicular epithelium (IFE) in μm.sup.2, an     their mean cortimedullary ratios (C/M) from 20 chickens.                      .sup.2 Means which do not share a superscript differ significantly at p       ≦ 0.05 by analysis of variance, using the Tukey test to identify       significant differences.                                                 

                  TABLE I                                                         ______________________________________                                        Morphometry of Lymphoid Follicles                                             One Week after Treatment.sup.1, 2                                             Treatment                                                                              Follicle Medulla  IFE    Cortex C/M                                  ______________________________________                                        None     64,600   11,900.sup.a                                                                           21,000.sup.a                                                                         31,700 2.7.sup.a                            Vehicle  53,800    8,870   16,000.sup.a, b                                                                      28,400 3.3.sup.a, d                         1.0 mg   56,800    7,190   14,300.sup.b                                                                         35,400 5.4.sup.b                            2.5 mg   54,500    7,200   14,500.sup.b                                                                         32,900 4.8.sup.b, c                         5.0 mg   62,500    9,650   16,900.sup.a, b                                                                      35,900 3.8.sup.c, d                         ______________________________________                                         .sup.1 Values are mean areas of 25 contiguous follicles, their medullae,      cortices and zones of interfollicular epithelium (IFE) in μm.sup.2, an     their mean cortimedullary ratios (C/M) from 20 chickens.                      .sup.2 Means which do not share a superscript differ significantly at p       ≦ 0.05 by analysis of variance, using the Tukey test to identify       significant differences.                                                 

                  TABLE J                                                         ______________________________________                                        Morphometry of Lymphoid Follicles                                             Two Weeks after Treatment.sup.1, 2                                            Treatment                                                                             Follicle  Medulla  IFE    Cortex C/M                                  ______________________________________                                        None    228,000.sup.a, c                                                                        42,600.sup.a                                                                           65,200.sup.a                                                                         120,000                                                                              2.9.sup.a                            Vehicle 213,000.sup.a, c                                                                        39,800.sup.a                                                                           57,400.sup.a                                                                         116,000                                                                              3.1.sup.a                            1.0 mg  170,000.sup.a                                                                           19,300.sup.b, c                                                                        43,700.sup.b                                                                         107,000                                                                              5.4.sup.b                            2.5 mg  274,000.sup.b, c                                                                        35,900.sup.a, c                                                                        65,400.sup.a                                                                         173,000                                                                              5.0.sup.b                            5.0 mg  242,000.sup.a, c                                                                        43,600.sup.a                                                                           65,400.sup.a                                                                         133,000                                                                              3.3.sup.a                            ______________________________________                                         .sup.1 Values are mean areas of 25 contiguous follicles, their medullae,      cortices and zones of interfollicular epithelium (IFE) in μm.sup.2, an     their mean cortimedullary ratios (C/M) from 20 chickens.                      .sup.2 Means which do not share a superscript differ significantly at p       ≦ 0.05 by analysis of variance, using the Tukey test to identify       significant differences.                                                 

                  TABLE K                                                         ______________________________________                                        Morphometry of Lymphoid Follicles                                             Three Weeks after Treatment.sup.1, 2                                          Treatment                                                                             Follicle  Medulla  IFE    Cortex C/M                                  ______________________________________                                        None    436,000   72,900   102,000                                                                              256,000                                                                              3.7.sup.a                            Vehicle 429,000   74,500   118,000                                                                              236,000                                                                              3.3.sup.a                            1.0 mg  500,000   76,600   129,000                                                                              295,000                                                                              4.0.sup.a                            2.5 mg  527,000   75,500   134,000                                                                              302,000                                                                              4.2.sup.b                            5.0 mg  478,000   74,800   132,000                                                                              272,000                                                                              3.8.sup.a, b                         ______________________________________                                         .sup.1 Values are mean areas of 25 contiguous follicles, their medullae,      cortices and zones of interfollicular epithelium (IFE) in μm.sup.2, an     their mean cortimedullary ratios (C/M) from 20 chickens.                      .sup.2 Means which do not share a superscript differ significantly at p       ≦ 0.05 by analysis of variance, using the Tukey test to identify       significant differences.                                                 

7.19 Immunostimulation in Poultry

Chickens treated with 1.0 mg of the copolymer T150R1 in an oil-in-wateremulsion at one day of age are immunized at one and two weeks with sheepred blood cells (SRBC) and killed Brucella abortus (BA) to elicitprimary and secondary antibody responses, as described by the method ofBhanushali et al., (See Bhanushali, J. K., K. K. Murthy and W. L.Ragland, The effects of in ovo mibolerone treatment on the bursa ofFabricius and the humoral immune system of chickens: A dose-responsestudy. Immunopharmacology 10: 99-110, 1985) which is incorporated byreference herein. Results of an experiment with 20 chickens per groupare shown in Table L. Most of the birds demonstrate no measurableprimary response to either antigen. The total Ig response, but not theIgG secondary response, to sheep red blood cells is significantly higherin chickens which have been treated with a dose containing 1.0 mg T150R1than in the other three groups. Secondary responses to Brucella abortusin treated birds are not different from birds injected with the vehiclealone.

In a subsequent immunization experiment, 40 chickens are treated with1.0 mg T150R1 and 40 with the vehicle. The data are shown in Table M.The total secondary Ig responses to both Brucella abortus and sheep redblood cells are significantly increased but there is no significanteffect on class switching to IgG, i.e., the effect is only on IgM.

                                      TABLE L                                     __________________________________________________________________________    Antibody Responses to Sheep Red Blood Cells (SRBC)                            and Killed Brucella abortus (BA) for 20 Chickens                                     SRBC Titers.sup.a                                                                            BA Titers.sup.a                                                     Secondary      Secondary                                          Treatment                                                                            Primary                                                                            -2 ME                                                                              +2 ME                                                                              Primary                                                                            -2 ME                                                                              +2 ME                                         __________________________________________________________________________    Vehicle                                                                              0.21 4.42 1.53 0.89 4.95 0.74                                          1.0 mg 0.22 .sup. 6.00.sup.b                                                                   2.22 0.83 5.11 0.83                                          2.5 mg 0.72 5.47 2.00 1.17 4.72 0.94                                          5.0 mg 0.12 5.59 1.76 0.89 4.95 0.74                                          __________________________________________________________________________     .sup.a Total immunoglobulin titers are measured with untreated serum (-2      ME) and IgG titers with serum to which 2mercaptothanol has been added (+2     ME). Titer is determined by log.sub.2 of the reciprocal of the highest        serum dilution to cause agglutination.                                        .sup.b Significantly different from vehicle control, p ≦ 0.05     

                                      TABLE M                                     __________________________________________________________________________    Antibody Responses to Sheep Red Blood Cells (SRBC)                            and Killed Brucella abortus (BA) for 40 Chickens                                     SRBC Titers.sup.a                                                                            BA Titers.sup.a                                                     Secondary      Secondary                                          Treatment                                                                            Primary                                                                            -2 ME                                                                              +2 ME                                                                              Primary                                                                            -2 ME                                                                              +2 ME                                         __________________________________________________________________________    Vehicle                                                                              0.69 4.46 0.66 0.45 5.45 0.90                                          1.0 mg 0.97 .sup. 5.20.sup.b                                                                   0.69 0.84 .sup. 6.23.sup.b                                                                   0.83                                          __________________________________________________________________________     .sup.a Total immunoglobulin titers are measured with untreated serum (-2      ME) and IgG titers with serum to which 2mercaplvethanal has been added (+     ME). Titer was determined by log.sub.2 of the reciprocal of the highest       serum dilution to cause agglutination.                                        .sup.b Significantly different from vehicle control, p ≦ 0.05.    

In the first above-described experiment involving immunized chickens,heparinized blood is collected and buffy coats are prepared forblastogenic tests. Proliferation of lymphocytes stimulated withphytahemagglutinin and concanavalin A are not different from vehiclecontrols suggesting that the polymer has little effect in cell-mediatedresponses in chickens. Data are not shown.

It should be understood, of course, that the foregoing relates only to apreferred embodiment of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

We claim:
 1. A method of stimulating the growth of animals comprisingthe step of:administering to an animal an amount of abiologically-active copolymer sufficient to stimulate the growth of theanimals, the biologically-active copolymer comprising an octablockcopolymer having the following formula: ##STR14## wherein: the meanaggregate molecular weight of the portion of the octablock copolymerrepresented by polyoxypropylene is between approximately 5000 and 7000daltons;a is a number such that the portion represented bypolyoxyethylene constitutes between approximately 10% to 40% of thecompound by weight, and; b is a number such that the polyoxypropyleneportion of the total molecular weight of the octablock copolymerconstitutes between approximately 60% and 90% of the compound by weight.2. The method of claim 1, wherein said octablock copolymer comprises acompound with the following formula: ##STR15## wherein: the meanaggregate molecular weight of the portion of the octablock copolymerrepresented by polyoxypropylene is approximately 6750 daltons;a is anumber such that the portion of the total molecular weight representedby polyoxyethylene constitutes approximately 10% of the compound byweight; and b is a number such that the polyoxypropylene portion of theoctablock copolymer constitutes approximately 90% of the compound byweight.
 3. The method of claim 1, wherein the biologically activecopolymer is injected in an oil and water emulsion.
 4. The method ofclaim 1 wherein the ratio of oil to water in said oil and water emulsionis between approximately 80:20 and 1:100.
 5. The method of claim 1wherein the octablock copolymer is administered by injection.
 6. Themethod of claim 1 wherein the octablock copolymer is administeredorally.
 7. The method of claim 1, wherein said octablock copolymercomprises a compound with the following formula: ##STR16## wherein: themean aggregate molecular weight of the portion of the octablockcopolymer represented by polyoxypropylene is approximately 5750daltons;a is a number such that the portion of the total molecularweight represented by polyoxyethylene constitutes approximately 10% ofthe compound by weight; and b is a number such that the polyoxypropyleneportion of the octablock copolymer constitutes approximately 90% of thecompound by weight.
 8. The method of claim 1 wherein the octablockcopolymer is administered orally with food.
 9. The method of claim 1wherein the octablock copolymer is administered with water.